Dosages and methods for delivering lipid formulated nucleic acid molecules

ABSTRACT

Methods, kits and devices for dosing a subject to reduce a hypersensitivy response to a lipid-formulated nucleic acid (e.g., RNA) molecule are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/889,352, filed Nov. 5, 2015, now allowed, which is a U.S. NationalPhase Application under 35 U.S.C. § 371 of International Application No.PCT/US2014/036915, filed May 6, 2014, which claims the benefit of U.S.Provisional Application No. 61/820,036, filed May 6, 2013. The contentsof the aforesaid applications are hereby incorporated by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 2, 2014, isnamed Sequence_Listing.txt and is 1,785 bytes in size.

BACKGROUND OF THE INVENTION

Infusion-related reactions (IRRs) relate to any signs or symptomsexperienced by a subject during infusion of a pharmacological orbiological agent (e.g., a drug). These reactions can be acute, andtypically occur during the first hour(s) or day after drugadministration. Acute infusion-related reactions include a variety ofsigns and symptoms, including, but not limited to, neurologic (e.g.,dizziness, headache, weakness, syncope, seizure), psychiatric (e.g.,anxiety), cardiovascular (e.g., tachycardia, hypotension, arrhythmia,chest pain, ischemia or infarction, cardiac arrest), cutaneous (e.g.,flushing, erythema, pruritus, urticaria, angioedema, maculopapularrash), and gastrointestinal signs and symptoms. Manifestations of IRRscan vary, and can include hypersensitivity reactions.

Drug hypersensitivity often results from interactions between apharmacologic agent and the immune system. Identifiable risk factors fordrug hypersensitivity reactions include age, female gender, concurrentillnesses, and previous hypersensitivity to related drugs. Drughypersensitivity reactions can be classified into immune mediated andnon-immune mediated hypersensitivity reactions. Immune mediatedhypersensitivity reactions are typically associated with specific,adaptive immune responses generated against an antigen, e.g., a drug.These reactions can occur in a sensitized patient, and can be classifiedinto the following types: Type 1 or immediate IgE-mediated response;Type 2 or antibody dependent cytotoxicity; Type 3 or Immune complexmediated; and Type 4 or delayed response (T cell-mediated) (reviewed in“Drug Hypersensitivity Reactions: Risk Assessment and Management,Society for Toxicology Course” (2011)). Non-immune mediatedhypersensitivity reactions relate to drug responses initiated by somepharmacological action of the drug; these reactions can involve immunesystem components.

Non-immune mediated hypersensitivity reactions, also known aspseudoallergic or anaphylactoid reactions, have clinical manifestationsthat are often indistinguishable from allergic reactions. Thesereactions are believed to be associated with complement activation, aswell as degranulation of mast cells and/or basophils, which in turncauses histamine release and an anaphylactic-like reaction. Non-immunemediated hypersensitivity reactions are not believed to be triggered viaIgE and Fc epsilon receptor activation, and no-presensitization isnecessary for a response to occur.

Thus, the need exists for developing novel methods and compositions thatreduce hypersensitivity reactions to drugs.

SUMMARY OF THE INVENTION

Disclosed herein are methods, kits and devices for dosing a subject toreduce an infusion-related reaction (IRR) and/or a hypersensitivyreaction (e.g., to reduce the incidence and/or severity of an IRR or ahypersensitivity reaction) to a lipid-formulated nucleic acid (e.g.,RNA, e.g., a siRNA) molecule. Without wishing to be bound by theory,Applicants have discovered that infusion reactions to compositions thatinclude a lipid formulation and a nucleic acid (e.g., RNA, e.g., asiRNA) molecule tend to be associated with an IRR and/orhypersensitivity reactions, for example, non-immune mediatedhypersensitivity reactions (also referred to herein as pseudoallergicreactions). In one embodiment, administration of a first dose (or apre-dose) of a lipid-formulated RNA molecule corresponding to a portionof a second dose or the total dose, or administration of a first dose ata portion of the rate of infusion of a second dose, over a pre-treatmentinterval, was found to reduce or prevent the IRR or the hypersensitivityreaction in a subject. Thus, methods, kits and devices for dosing asubject to reduce an IRR or a hypersensitivy reaction to alipid-formulated nucleic acid molecule are disclosed.

Accordingly, in one aspect, the invention features a method of reducingan infusion-related reaction, or a hypersensitivity reaction, or both,in a subject, to a composition comprising a lipid formulation and anucleic acid molecule (e.g., an RNA molecule capable of mediating RNAinterference). The method includes administering to a subject a firstdose and a second dose of said composition. In certain embodiments, themethod includes one, two, three, four, five, six, seven or all of a)-h)of the following:

a) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or50, of the amount of said composition administered in said second dose;

b) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40or 50, of the total amount of said composition administered;

c) the first dose is administered over a time period that is no morethan 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time periodover which the second dose is administered;

d) the first dose is administered over a time period that is no morethan 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time periodover which the total dose is administered;

e) the rate of administration, e.g., in mg/min or mL/min, of said firstdose is no more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40 or 50, of the rate of administration of said second dose, orthe rate of administration of the total dose;

f) the amount of said composition administered in said first dose is nomore than 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg nucleicacids per kg body weight, and the second dose is greater than said firstdose;

g) the amount of said composition administered in said second dose isgreater than 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000 μg nucleic acids per kg body weight, and the second dose is greaterthan said first dose; or

h) the dosages and time periods of administration of said first andsecond doses are selected such that no substantial IRR and/orhypersensitivity reaction occurs in said subject.

In some embodiments, the method includes one, two, three, four, five,six, seven or all of a)-h) of the following:

a) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 9, 10 or 15, e.g., 10, of the amount of saidcomposition administered in said second dose;

b) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 9, 10 or 15, e.g., 10, of the total amountof said composition administered;

c) the first dose is administered over a time period that is no morethan 1/X, wherein X is 2, 3 or 4, e.g., 3, of the time period over whichthe second dose is administered;

d) the first dose is administered over a time period that is no morethan 1/X, wherein X is 3, 4 or 5, e.g., 4, of the time period over whichthe total dose is administered;

e) the rate of administration, e.g., in mg/min or mL/min, of said firstdose is no more than 1/X, wherein X is 2, 3 or 4, e.g., 3, of the rateof administration of said second dose, or the rate of administration ofthe total dose;

f) the amount of said composition administered in said first dose is nomore than 20, 30 or 40 μg, e.g., 30 μg, nucleic acids per kg bodyweight, and the second dose is greater than said first dose;

g) the amount of said composition administered in said second dose isgreater than 100, 200 or 300 μg, e.g., 200 μg, nucleic acids per kg bodyweight, and the second dose is greater than said first dose; or

h) the dosages and time periods of administration of said first andsecond doses are selected such that no substantial IRR and/orhypersensitivity reaction occurs in said subject.

In another aspect, the invention features a method of reducing theexpression of a target gene, or treating a disorder related to thetarget gene, in a subject. The method includes administering to thesubject a first dose and a second dose of a composition, saidcomposition comprising a lipid formulation and a nucleic acid molecule(e.g., an RNA molecule capable of mediating RNA interference), whereinsaid first and second doses are administered in an amount sufficient toreduce expression of the target gene, or treat the disorder, in thesubject. In certain embodiments, the method includes one, two, three,four, five, six or all of a)-g) of the following:

a) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or50, of the amount of said composition administered in said second dose;

b) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40or 50, of the total amount of said composition administered;

c) the first dose is administered over a time period that is no morethan 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time periodover which the second dose is administered;

d) the first dose is administered over a time period that is no morethan 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time periodover which the total dose is administered;

e) the rate of administration, e.g., in mg/min or mL/min, of said firstdose is no more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40 or 50, of the rate of administration of said second dose, orthe total dose;

f) the amount of said composition administered in said first dose is nomore than 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg nucleicacids per kg body weight and said first dose is less than said seconddose; or

g) the amount of said composition administered in said second dose isgreater than 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000 μg nucleic acids per kg body weight, and said second dose isgreater than said first dose.

In some embodiments, the method includes one, two, three, four, five,six or all of a)-g) of the following:

a) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 9, 10 or 15, e.g., 10, of the amount of saidcomposition administered in said second dose;

b) the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 9, 10 or 15, e.g., 10, of the total amountof said composition administered;

c) the first dose is administered over a time period that is no morethan 1/X, wherein X is 2, 3 or 4, e.g., 3, of the time period over whichthe second dose is administered;

d) the first dose is administered over a time period that is no morethan 1/X, wherein X is 3, 4 or 5, e.g., 4, of the time period over whichthe total dose is administered;

e) the rate of administration, e.g., in mg/min or mL/min, of said firstdose is no more than 1/X, wherein X is 2, 3 or 4, e.g., 3, of the rateof administration of said second dose, or the rate of administration ofthe total dose;

f) the amount of said composition administered in said first dose is nomore than 20, 30 or 40 μg, e.g., 30 μg, nucleic acids per kg bodyweight, and the second dose is greater than said first dose; or

g) the amount of said composition administered in said second dose isgreater than 100, 200 or 300 μg, e.g., 200 μg, nucleic acids per kg bodyweight, and the second dose is greater than said first dose.

Exemplary Determination of First and Second Doses and Rates ofAdministration

The amount, dose and rate of administration can be calculated based onthe lipid content of the composition (also referred to herein as the“lipid” amount, dose or dose rate; or based on the RNA molecule contentof the composition (referred to herein as “RNA,” “iRNA” or “siRNA”amount, dose or dose rate).

In one embodiment, in the microdosing regimen, 1/X of the total dose D(μg/kg) is administered over a first time period T₁ (min) and (X-1)/X ofthe same total dose is administered over a second time period T₂ (min)in a subject having a body weight of W (kg). Accordingly, the first doseis administered at a dose rate of D/X/T₁ (μg/kg/min) (or W*D/X/T₁(μg/min)) and the second dose is administered at a dose rate ofD/T₂-D/X/T₂ (μg/kg/min) (or W*(D/T₂−D/X/T₂) (μg/min)). In one exemplaryembodiment, the first and second dose rates are obtained as follows: Byadministering a first dose (e.g., microdosing) of 1/10^(th) of the totaldose over a first time interval (e.g., 15 minutes), followed by a seconddose of 9/10^(th) of total dose over a second time interval (e.g., 60minutes), in a subject having a body weight of 80 kg. For example, forthe first dose rate, 1/10 of the total siRNA dose 300 μg/kg (i.e., 30μg/kg) is administered over a first time period of 15 minutes, thusresulting in a first dose rate of 2 μg/kg/min (or 160 μg/min) (iRNA doserate). The remaining dose or second dose ((10-1)/10= 9/10 of the totaliRNA dose of 300 μg/kg=270 μg/kg) is administered over 60 min, thusresulting in a second dose rate of 4.5 μg/kg/min (or 360 μg/min). Inorder to calculate a lipid dose rate, we assumed a total lipid/siRNAratio of 12.23:1 (wt/wt) for conversion. In such embodiments, the firstdose is administered at a lipid dose rate of about 24 μg/kg/min (or 1920μg/min) (or at an iRNA dose rate of about 2 μg/kg/min) over a period ofabout 15 minutes, and the second dose is administered at a lipid doserate of about 55 μg/kg/min (or 4400 μg/min) (or at an siRNA dose rate ofabout 4.5 μg/kg/min) over a period of about 60 minutes.

Values for first and second dose rates can be obtained for other totaldoses applying the criteria set for above. In one embodiment, the firstand second doses are obtained from a composition formulated as an LNPformulation (e.g., an LNP11 formulation as described herein), which isadministered at a total dose range of about 0.05 to about 5 μg/kg (e.g.,10, 50, 150 and 300 microgram/kg) of iRNA. The range of total lipid/iRNAratios is from about 5 to about 30, e.g., about 8 to about 20, or about10 to about 15.

Applying the same approach to other doses (e.g., 10, 50, and 150microgram/kg) of iRNA, the following values for first and second doserates are obtained: 0.07 μg/kg/min (first iRNA dose rate) and 0.15μg/kg/min (second iRNA dose rate) for 10 μg/kg dose of iRNA, 0.33μg/kg/min (first iRNA dose rate) and 0.75 μg/kg/min (second iRNA doserate) for 50 μg/kg dose of iRNA, 1 μg/kg/min (first iRNA dose rate) and2 μg/kg/min (second iRNA dose rate) for 150 μg/kg dose of iRNA. The doserates described above can be converted to μg/min based on the bodyweight of the subject using the calculation methods described herein.

Alternatively, or in combination with the exemplary embodiment above,microdosing at 1/10^(th) of the non-microdosing infusion rate (e.g.,1/10^(th) of the 60 min infusion rate for 15 min (first dose) followedby the remainder of the dose at the non-microdosing 60 min infusion rate(second dose, total dosing time depends on the weight of the subject).

For example, in the non-microdosing regimen, a total dose of D (μg/kg)is administered at a dose rate R (μg/kg/min) over a total time period ofT (min). Accordingly, R=D/T. In the corresponding microdosing regimen,the first dose is administered at 1/X of the dose rate R (μg/kg/min)over a first time period T₁ (min) and the remaining dose is administeredat the dose rate R over a second time period T₂ (min). Accordingly,T₂=T−T₁/X. For example, in the non-microdosing regimen, a total siRNAdose of 300 (μg/kg) is administered at an iRNA dose rate of 300/60=5μg/kg/min over a total time period of 60 minutes. In the correspondingmicrodosing regimen, the first dose is administered at 1/10 of the iRNAdose rate 5 μg/kg/min=0.5 μg/kg/min over a first time period 15 minutesand the remaining dose is administered at the siRNA dose rate of 5μg/kg/min over a second time period 60-15/10=58.5 minutes. In thisexemplary calculation, the total lipid/siRNA ratio of 12.23 (wt/wt) isused for conversion. In such embodiments, the first dose is administeredat a lipid dose rate of about 6.1 μg/kg/min (or at an iRNA dose rate ofabout 0.5 μg/kg/min) over a period of about 15 minutes, and the seconddose is administered at a lipid dose rate of about 61 μg/kg/min (or atan iRNA dose rate of about 5 μg/kg/min) over a period of about 60minutes.

Values for first and second dose rates can be obtained for other totaldoses applying the criteria set for above. In one embodiment, the firstand second doses are obtained from a composition formulated as an LNPformulation (e.g., an LNP11 formulation as described herein), which isadministered at a total dose range of about 0.05 to about 5 μg/kg (e.g.,10, 50, 150 and 300 microgram/kg) of iRNA. The range of total lipid/iRNAratios is from about 5 to about 30, e.g., about 8 to about 20, or about10 to about 15.

Applying the same approach to other doses (e.g., 10, 50, and 150microgram/kg) of iRNA, the following values for first and second doserates are obtained: 0.017 μg/kg/min (first iRNA dose rate) and 0.17μg/kg/min (second iRNA dose rate) for 10 μg/kg dose of iRNA, 0.083μg/kg/min (first iRNA dose rate) and 0.83 μg/kg/min (second iRNA doserate) for 50 μg/kg dose of iRNA, 0.25 μg/kg/min (first iRNA dose rate)and 2.5 μg/kg/min (second iRNA dose rate) for 150 μg/kg dose of iRNA.The dose rates described above can be converted to μg/min based on thebody weight of the subject using the calculation methods describedherein.

As another example, in the non-microdosing regimen, a total dose of D(μg/kg) is administered at a dose rate R (μg/min) over a total timeperiod of T (min) in a subject having a body weight of W (kg). In thecorresponding microdosing regimen, the first dose is administered at 1/Xof the dose rate R (μg/min) over a first time period T₁ (min) and theremaining dose (W*D-T₁*R/X (μg)) is administered at the dose rate R overa second time period T₂ (min). Accordingly, T₂=W*D/R−T₁/X. For example,in the non-microdosing regimen, a total siRNA dose of 300 (μg/kg) isadministered at an iRNA dose rate of 400 μg/min over a total time periodof 60 minutes in a subject having a body weight of 80 kg. In thecorresponding microdosing regimen, the first dose is administered at1/10 of the iRNA dose rate 400 μg/min=40 μg/min over a first time period15 minutes and the remaining dose (80 (kg)×300 (μg/kg)−40 (μg/min)×15(min)=23400 μg) is administered at the siRNA dose rate of 400 μg/minover a second time period 23400 (μg)/400 (μg/min)=58.5 min. The totallipid/siRNA ratio of 12.23 (wt/wt) is used for conversion. In suchembodiments, the first dose is administered at a lipid dose rate ofabout 489.2 μg/min (or at an iRNA dose rate of about 40 μg/min) over aperiod of about 15 minutes, and the second dose is administered at alipid dose rate of about 4892 μg/min (or at an iRNA dose rate of about40 μg/min) over a period of about 60 minutes.

Values for first and second dose rates can be obtained for other totaldoses applying the criteria set for above. In one embodiment, the firstand second doses are obtained from a composition formulated as an LNPformulation (e.g., an LNP11 formulation as described herein), which isadministered at a total dose range of about 0.05 to about 5 μg/kg (e.g.,10, 50, 150 and 300 microgram/kg) of iRNA. The range of total lipid/iRNAratios is from about 5 to about 30, e.g., about 8 to about 20, or about10 to about 15.

Applying the same approach to other doses (e.g., 10, 50, and 150microgram/kg) of iRNA, the following values for first and second doserates are obtained for a subject having a body weight of 80 kg: 1.3μg/min (first iRNA dose rate) and 13.3 μg/min (second iRNA dose rate)for 10 μg/kg dose of iRNA, 6.7 μg/min (first iRNA dose rate) and 66.7μg/min (second iRNA dose rate) for 50 μg/kg dose of iRNA, 20 μg/min(first iRNA dose rate) and 200 μg/min (second iRNA dose rate) for 150μg/kg dose of iRNA.

As yet another example, in the non-microdosing regimen, a total dose of0.30 μg/kg of iRNA is administered at 3 mL/min over a period of 60minutes. In the corresponding microdosing regimen administered over aperiod of 70 minutes, a first dose is administered at 1 mL/min over aperiod of 15 minutes and a second dose is administered at 3 mL/min overa period of 55 minutes (a total dose of 0.30 μg/kg of iRNA in 180 mL).

The following values for the first and second iRNA dose rates areobtained for a subject: 1.67 μg/kg/min (first iRNA dose rate) and 5μg/kg/min (second iRNA dose rate). The following values for the firstand second lipid dose rates (lipid:siRNA ratio=11.6:1) are obtained fora subject: 19.4 μg/kg/min (first lipid dose rate) and 58 μg/kg/min(second lipid dose rate).

Based on the time intervals for administration, the following values forthe first and second iRNA doses are obtained for a subject: 25 μg/kg(first iRNA dose) and 275 μg/kg (second iRNA dose). Based on the timeintervals for administration, the following values for the first andsecond lipid doses (lipid:siRNA ratio=11.6:1) are obtained for asubject: 290 μg/kg (first lipid dose) and 3190 μg/kg (second lipiddose).

The dose rates and doses described above can be converted to mg/minbased on the body weight of the subject.

The following values for the first and second iRNA dose rates areobtained for a subject having a body weight of 70 kg: 0.117 mg/min(first iRNA dose rate) and 0.35 mg/min (second iRNA dose rate). Thefollowing values for the first and second lipid dose rates (lipid:siRNAratio=11.6:1) are obtained for a subject having a body weight of 70 kg:1.36 mg/min (first lipid dose rate) and 4.06 mg/min (second lipid doserate).

Based on the time intervals for administration, the following values forthe first and second iRNA doses are obtained for a subject having a bodyweight of 70 kg: 1.75 mg (first iRNA dose) and 19.25 mg (second iRNAdose). Based on the time intervals for administration, the followingvalues for the first and second lipid doses (lipid:siRNA ratio=11.6:1)are obtained for a subject having a body weight of 70 kg: 20.3 mg (firstlipid dose) and 223.3 mg (second lipid dose).

The same approach can be applied to any other total doses (e.g., 0.01,0.05, or 0.15 μg/kg) of iRNA, lipid:siRNA ratios (e.g., between 11.5 to14.1), body weight, and/or invervals for administration, e.g., asdescribed herein.

Other features and embodiments of the invention are described asfollows:

Doses

In certain embodiments, the amount of said composition administered insaid first dose is chosen from between (and including): about 1% toabout 25%, about 3% and about 20%, about 5% and about 15%, or about 8%and about 12% (e.g., about 9-10%), of the total amount of saidcomposition, or the amount of the composition administered in saidsecond dose.

In one embodiment, the amount of lipid formulation administered in saidfirst dose is chosen from about 5 μg/kg to about 3000 μg/kg, about 50μg/kg to about 2000 μg/kg, about 100 μg/kg to about 1500 μg/kg, about200 μg/kg to about 1000 μg/kg, or about 300 μg/kg to about 500 μg/kg.

Alternatively, or in combination with, the first dose values describedherein, the amount of lipid formulation administered in said second doseis chosen about 125 μg/kg to about 15000 μg/kg, about 500 μg/kg to about10000 μg/kg, about 1000 μg/kg to about 7500 μg/kg, about 2000 μg/kg toabout 5000 μg/kg, or about 3000 μg/kg to about 4000 μg/kg.

In yet other embodiments, the amount of the nucleic acid (e.g., RNA)molecule in said first dose is chosen from about 0.1 μg/kg to about 100μg/kg, about 0.5 μg/kg to about 75 μg/kg, about 1 μg/kg to about 50μg/kg, about 2 μg/kg to about 30 μg/kg, about 5 μg/kg to about 15 μg/kg.

Alternatively, or in combination with, the first dose values describedherein, the amount of the RNA molecule in said second dose is chosenfrom about 5 μg/kg to about 1000 μg/kg, about 25 μg/kg to about 800μg/kg, about 50 μg/kg to about 600 μg/kg, about 100 μg/kg to about 400μg/kg, or about 200 μg/kg to about 300 μg/kg.

In some embodiments, the total amount of the RNA molecule in said firstdose and said second dose is chosen from about 100 μg/kg to about 500μg/kg, or about 200 μg/kg to about 400 μg/kg, e.g., about 300 μg/kg.

In some embodiments, the body weight of the subject is chosen from about50 kg to about 150 kg, from about 50 kg to about 104 kg, from about 60kg to about 100 kg, from about 70 kg to about 90 kg, e.g, about 80 kg.

It shall be understood that the values provided herein for the doseranges for the first and second doses based on the composition, lipidformulation and/or RNA molecules can be combined in any order in themethods, kits and devices described herein. In certain embodiments, anyof the aforesaid values for the dose ranges can be combined with theadmininstration rates and time intervals for administration describedherein.

Dose Rates

In other embodiments, the rate of administration, e.g., in mg/min ormL/min, of said first dose is no more than 1/X, wherein X is 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40 or 50, of the rate of administration of saidsecond dose, or the rate of administration of the total dose. In oneembodiment, the rate of administration, e.g., in mg/min or mL/min, ofsaid first dose is chosen from one or more of: about 5% to 50%, about 5%to about 20%, about 5% to 10%, about 8% to 45%, about 10% and 40%, about10% to about 20%, or about 10% and 35%, or about 20% to about 40% (e.g.,about 10-33%) of the rate of administration of the second dose, or thetotal dose.

In yet other embodiments, the rate of administration of the lipidformulation in said first dose is chosen from about 0.05 μg/min/kg toabout 50 μg/min/kg, about 0.1 μg/min/kg to about 25 μg/min/kg, about 1μg/min/kg to about 15 μg/min/kg, or about 5 μg/min/kg to about 10μg/min/kg. In yet other embodiments, the rate of administration of thelipid formulation in said first dose is chosen from about 2.5 μg/min toabout 5000 μg/min, about 5 μg/min to about 2500 μg/min, about 50 μg/minto about 1500 μg/min, or about 250 μg/min to about 1000 μg/min.

Alternatively, or in combination with, the rate of administration valuesfor the first dose described herein, the rate of administration of thelipid formulation in said second dose is chosen from about 0.5 μg/min/kgto about 500 μg/min/kg, about 1 μg/min/kg to about 250 μg/min/kg, about10 μg/min/kg to about 150 μg/min/kg, or about 50 μg/min/kg to about 100μg/min/kg. In yet other embodiments, the rate of administration of thelipid formulation in said second dose is chosen from about 25 μg/min toabout 50000 μg/min, about 50 μg/min to about 25000 μg/min, about 500μg/min to about 15000 μg/min, or about 2500 μg/min to about 10000μg/min.

In yet other embodiments, the rate of administration of the nucleic acid(e.g., RNA) molecule in said first dose is chosen from about 0.01μg/min/kg to about 5 μg/min/kg, about 0.02 μg/min/kg to about 2.5μg/min/kg, about 0.05 μg/min/kg to about 1 μg/min/kg, or about 0.1μg/min/kg to about 0.5 μg/min/kg. In yet other embodiments, the rate ofadministration of the nucleic acid (e.g., RNA) molecule in said firstdose is chosen from about 0.5 μg/min to about 600 μg/min, about 1 μg/minto about 250 μg/min, about 2.5 μg/min to about 100 μg/min, or about 5μg/min to about 50 μg/min.

Alternatively, or in combination with, the rate of administration valuesfor the first dose described herein, the rate of administration of thenucleic acid (e.g., RNA) molecule in said second dose is chosen fromabout 0.1 μg/min/kg to about 50 μg/min/kg, about 0.2 μg/min/kg to about25 μg/min/kg, about 0.5 μg/min/kg to about 10 μg/min/kg, or about 1μg/min/kg to about 5 μg/min/kg. In yet other embodiments, the rate ofadministration of the nucleic acid (e.g., RNA) molecule in said seconddose is chosen from about 5 μg/min to about 6000 μg/min, about 10 μg/minto about 2500 μg/min, about 25 μg/min to about 1000 μg/min, or about 50μg/min to about 500 μg/min.

In yet other embodiments, the rate of administration in said first doseis chosen from about 0.5 mL/min to about 2 mL/min, e.g., about 1 mL/min.

Alternatively, or in combination with, the rate of administration valuesfor the first dose described herein, the rate of administration in saidsecond dose is chosen from about 2 mL/min to about 4 mL/min, e.g., about3 mL/min.

It shall be understood that the values provided herein for the rates ofadministration for the first and second doses based on the composition,lipid formulation and/or RNA molecules can be combined in any order inthe methods, kits and devices described herein. In certain embodiments,any of the aforesaid values for the rates of administration can becombined with the dose ranges and time intervals for administrationdescribed herein.

For example, the first and second rate of administration values can becombined as follows:

i) the rate of administration of the lipid formulation in said firstdose is chosen from about 0.05 μg/min/kg to about 50 μg/min/kg, about0.1 μg/min/kg to about 25 μg/min/kg, about 1 μg/min/kg to about 15μg/min/kg, or about 5 μg/min/kg to about 10 μg/min/kg; and

ii) the rate of administration of the lipid formulation in said seconddose is chosen from about 0.5 μg/min/kg to about 500 μg/min/kg, about 1μg/min/kg to about 250 μg/min/kg, about 10 μg/min/kg to about 150μg/min/kg, or about 50 μg/min/kg to about 100 μg/min/kg.

As another example, the first and second rate of administration valuescan be combined as follows:

i) the rate of administration of the lipid formulation in said firstdose is chosen from about 2.5 μg/min to about 5000 μg/min, about 5μg/min to about 2500 μg/min, about 50 μg/min to about 1500 μg/min, orabout 250 μg/min to about 1000 μg/min; and

ii) the rate of administration of the lipid formulation in said seconddose is chosen from about 25 μg/min to about 50000 μg/min, about 50μg/min to about 25000 μg/min, about 500 μg/min to about 15000 μg/min, orabout 2500 μg/min to about 10000 μg/min.

As yet another example, the first and second rate of administrationvalues can be combined as follows:

i) the rate of administration in said first dose is chosen from about0.5 mL/min to about 2 mL/min, e.g., about 1 mL/min; and

ii) the rate of administration in said second dose is chosen from about2 mL/min to about 4 mL/min, e.g., about 3 mL/min.

In yet other embodiments,

i) the rate of administration of the lipid formulation in said firstdose is chosen from about 0.05 μg/min/kg to about 50 μg/min/kg, about0.1 μg/min/kg to about 25 μg/min/kg, about 1 μg/min/kg to about 15μg/min/kg, or about 5 μg/min/kg to about 10 μg/min/kg; and

ii) the rate of administration of the lipid formulation in said seconddose is chosen from about 0.5 μg/min/kg to about 500 μg/min/kg, about 1μg/min/kg to about 250 μg/min/kg, about 10 μg/min/kg to about 150μg/min/kg, or about 50 μg/min/kg to about 100 μg/min/kg; and

wherein the first dose is administered over a time period that is nogreater than 1/X, wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., X is 3,4, or 5, e.g., 4) the time period over which the total dose isadministered.

In yet other embodiments,

i) the rate of administration of the lipid formulation in said firstdose is chosen from about 2.5 μg/min to about 5000 μg/min, about 5μg/min to about 2500 μg/min, about 50 μg/min to about 1500 μg/min, orabout 250 μg/min to about 1000 μg/min; and

ii) the rate of administration of the lipid formulation in said seconddose is chosen from about 25 μg/min to about 50000 μg/min, about 50μg/min to about 25000 μg/min, about 500 μg/min to about 15000 μg/min, orabout 2500 μg/min to about 10000 μg/min.

wherein the first dose is administered over a time period that is nogreater than 1/X, wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., X is 3,4, or 5, e.g., 4) the time period over which the total dose isadministered.

In other embodiments, the first and second rate of administration valuescan be combined as follows:

(i) the rate of administration of the nucleic acid (e.g., RNA) moleculein said first dose is chosen from about 0.01 μg/min/kg to about 5μg/min/kg, about 0.02 μg/min/kg to about 2.5 μg/min/kg, about 0.05μg/min/kg to about 1 μg/min/kg, or about 0.1 μg/min/kg to about 0.5μg/min/kg; and

(ii) the rate of administration of the nucleic acid (e.g., RNA) moleculein said second dose is chosen from about 0.1 μg/min/kg to about 50μg/min/kg, about 0.2 μg/min/kg to about 25 μg/min/kg, about 0.5μg/min/kg to about 10 μg/min/kg, or about 1 μg/min/kg to about 5μg/min/kg.

In other embodiments, the first and second rate of administration valuescan be combined as follows:

(i) the rate of administration of the nucleic acid (e.g., RNA) moleculein said first dose is chosen from about 0.5 μg/min to about 600 μg/min,about 1 μg/min to about 250 μg/min, about 2.5 μg/min to about 100μg/min, or about 5 μg/min to about 50 μg/min; and

(ii) the rate of administration of the nucleic acid (e.g., RNA) moleculein said second dose is chosen from about 5 μg/min to about 6000 μg/min,about 10 μg/min to about 2500 μg/min, about 25 μg/min to about 1000μg/min, or about 50 μg/min to about 500 μg/min.

In yet other embodiments, the first and second rate of administrationvalues can be combined as follows:

(i) the rate of administration of the nucleic acid (e.g., RNA) moleculein said first dose is chosen from about 0.01 μg/min/kg to about 5μg/min/kg, about 0.02 μg/min/kg to about 2.5 μg/min/kg, about 0.05μg/min/kg to about 1 μg/min/kg, or about 0.1 μg/min/kg to about 0.5μg/min/kg; and

(ii) the rate of administration of the nucleic acid (e.g., RNA) moleculein said second dose is chosen from about 0.1 μg/min/kg to about 50μg/min/kg, about 0.2 μg/min/kg to about 25 μg/min/kg, about 0.5μg/min/kg to about 10 μg/min/kg, or about 1 μg/min/kg to about 5μg/min/kg; and

wherein the first dose is administered over a time period that is nogreater than 1/X, wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., X is 3,4, or 5, e.g., 4) the time period over which the total dose isadministered.

In yet other embodiments, the first and second rate of administrationvalues can be combined as follows:

(i) the rate of administration of the nucleic acid (e.g., RNA) moleculein said first dose is chosen from about 0.5 μg/min to about 600 μg/min,about 1 μg/min to about 250 μg/min, about 2.5 μg/min to about 100μg/min, or about 5 μg/min to about 50 μg/min; and

(ii) the rate of administration of the nucleic acid (e.g., RNA) moleculein said second dose is chosen from about 5 μg/min to about 6000 μg/min,about 10 μg/min to about 2500 μg/min, about 25 μg/min to about 1000μg/min, or about 50 μg/min to about 500 μg/min; and

wherein the first dose is administered over a time period that is nogreater than 1/X, wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 the time periodover which the total dose is administered.

In yet other embodiments, the first and second rate of administrationvalues can be combined as follows:

i) the rate of administration in said first dose is chosen from about0.5 mL/min to about 2 mL/min, e.g., about 1 mL/min; and

ii) the rate of administration in said second dose is chosen from about2 mL/min to about 4 mL/min, e.g., about 3 mL/min; and

wherein the first dose is administered over a time period that is nogreater than 1/X, wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., X is 3,4, or 5, e.g., 4) the time period over which the total dose isadministered.

Time Interval for Administration

Alternatively, or in combination with, the dose ranges and rates ofadministration described herein, the first dose is administered over atime period that is no greater than 1/X, wherein X=2, 3, 4, 5, 6, 7, 8,9 or 10 times the time period over which the total dose is administered.

Alternatively or in combination with time interval for administration ofthe first dose described herein, the second dose is administered over atime period that is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times greaterthan the time period over which the first dose is administered.

In certain embodiments, the first dose is administered over a timeperiod that is between 5% and 50%, between 10% and 45%, between 15% and40%, between 20% and 35%, or between 25% and 30% of the time period ofadministration of the second dose (e.g., about 27% of the second timeperiod).

In other embodiments, the first dose is administered over a time periodthat is between 5 minutes and 60 minutes, between 10 minutes and 50minutes, between 20 minutes and 40 minutes, between 5 minutes and 30minutes, or between 10 minutes and 20 minutes (e.g., about 15 minutes).

Alternatively or in combination with time interval for administration ofthe first dose described herein, the second dose is administered over atime period that is between 30 minutes and 180 minutes, between 40minutes and 120 minutes, between 45 minutes and 90 minutes, or between50 minutes and 65 minutes (e.g., about 55 minutes).

In yet other embodiments, the first and second administration areeffected sequentially or substantially sequentially. In one embodiment,no more than 1, 10, 20, 30, 60, or 180 minutes separates the completionof the administration of the first dose and the initiation of theadministration of the second dose. In yet other embodiments, thecompletion of the administration of the first dose and the initiation ofthe administration of the second dose is essentially simultaneous.

In certain embodiments, the methods described herein further includeadministering to the subject one or more doses of the composition, e.g.,a third, fourth compositions.

Administration

The doses described herein can be administered by any suitable rout ofadministration, including but not limited to, intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion. In one embodiment, administration of the first and seconddoses is effected intravenously, e.g., by infusion (e.g., via a pump).In embodiments, the first and second doses are administered at asubstantially constant rate, e.g., via a pump or a sustained orcontrolled release formulation. In other embodiments, the first andsecond doses are administered as a gradient or multiple rates (e.g., twoor more rates of infusion).

In one embodiment, the flow rate of administration of the first dose isno more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40 or 50, of the flow rate of administration of said second dose. Forexample, the flow rate of administration of the first dose is chosenfrom about 0.5 to 1.5 mL/min, about 0.8 to 1.3 mL/min, or about 1 to 1.2mL/min (e.g., 1 mL/min or 1.1 mL/min); alternatively, or in combination,the flow rate of administration of the second dose is chosen from about2 to 4 mL/min, about 2.5 to 3.7 mL/min, or about 3 to 3.5 mL/min (e.g.,3 mL/min or 3.3 mL/min).

In certain embodiments, the total volume of infusion is about 100 to 300mL, about 150 to 250 mL, about 180 mL, or about 200 mL.

Methods of preparing the first and second dose as described herein arealso disclosed. For example, the methods can include the step ofmodifying the rate of administration of the composition, such that thedose is adjusted.

In an aspect provided herein, the compositions described arepharmaceutically acceptable, e.g., a pharmaceutical composition. Thepharmaceutical compositions can be administered in an unbufferedsolution, e.g., saline or water. In other embodiments, thepharmaceutical composition is administered with a buffer solution. Inembodiments, the buffer solution comprises acetate, citrate, prolamine,carbonate, or phosphate or any combination thereof. In embodiments, thebuffer solution is phosphate buffered saline (PBS).

In embodiments, the composition, e.g., pharmaceutical composition, isadministered intravenously.

In embodiments, the composition, e.g., pharmaceutical composition, isadministered subcutaneously.

Other features and embodiments of the invention include one or more ofthe following.

In certain embodiments of the aforesaid methods, the first dose isadministered at a first nucleic acid dose rate between 1.5 and 2μg/kg/min of, and the second dose is administered at a second nucleicacid dose rate between 4 and 6 μg/kg/min; and/or the first dose isadministered at a first lipid dose rate between 15 and 25 μg/kg/min, andthe second dose is administered at a second lipid dose rate between 55and 75 μg/kg/min. In other embodiments of the aforesaid methods, thefirst dose is administered at a first nucleic acid dose rate between 1.5and 2 μg/kg/min, and the second dose is administered at a second nucleicacid dose rate between 4 and 6 μg/kg/min;

In other embodiments of the aforesaid methods, the first lipid dose rateis between 15 and 25 μg/kg/min, and the second lipid dose rate isbetween 55 and 75 μg/kg/min.

In other embodiments of the aforesaid methods, the first dose isadministered at between 0.5 and 1.5 mL/min, and the second dose isadministered at between 2.5 and 3.5 mL/min. In other embodiments of theaforesaid methods, the first dose is administered over a period ofbetween 10 and 20 minutes, and the second dose is administered over aperiod of between 50 and 60 minutes.

In other embodiments of the aforesaid methods, the first nucleic aciddose is between 20 and 30 μg/kg, and the second nucleic acid dose isbetween 250 and 300 μg/kg. In other embodiments of the aforesaidmethods, the first lipid dose is between 250 and 400 μg/kg, and thesecond lipid dose is between 2500 and 4000 μg/kg.

In other embodiments of the aforesaid methods, the total nucleic aciddose in the first and the second doses is between 0.2 and 0.4 μg/kg,e.g., 0.3 μg/kg.

In other embodiments of the aforesaid methods, the total lipid dose inthe first and the second doses are between 3.0 and 4.5 μg/kg.

In other embodiments of the aforesaid methods, the first nucleic aciddose rate is between 0.1 and 0.15 mg/min, and the second nucleic aciddose rate is between 0.3 and 0.4 mg/min.

In other embodiments of the aforesaid methods, the first lipid dose rateis between 1.0 and 1.5 mg/min, and the second lipid dose rate is between3.5 and 4.5 mg/min.

In other embodiments of the aforesaid methods, the first nucleic aciddose is between 1.5 and 2.0 mg and the second nucleic acid dose isbetween 15 and 25 mg.

In other embodiments of the aforesaid methods, the first lipid dose isbetween 15 and 25 mg and the second lipid dose is between 200 and 300mg.

In another aspect, a method of reducing an infusion-related reaction, ora hypersensitivity reaction, or both, in a subject, to a compositioncomprising a lipid formulation, said lipid formulation comprising MC3and a siRNA molecule is provided. The method includes administering to asubject:

a first dose of said composition; and

a second dose of said composition;

wherein the first dose is administered at a first siRNA dose ratebetween 1.5 and 2 μg/kg/min of, and

wherein the second dose is administered at a second siRNA dose ratebetween 4 and 6 μg/kg/min; and/or

wherein the first dose is administered at a first lipid dose ratebetween 15 and 25 μg/kg/min, and

wherein the second dose is administered at a second lipid dose ratebetween 55 and 75 μg/kg/min.

In yet another aspect, a method of reducing the expression of a targetgene, or treating a disorder related to the target gene, in a subject,is provided. The method includes:

administering to the subject a first dose and a second dose of acomposition, said composition comprising a lipid formulation comprisingMC3 and an siRNA molecule, wherein said first and second doses areadministered in an amount sufficient to reduce expression of a targetgene, or treat the disorder, in the subject; and

wherein the first dose is administered at a first siRNA dose ratebetween 1.5 and 2 μg/kg/min of, and

wherein the second dose is administered at a second siRNA dose ratebetween 4 and 6 μg/kg/min; and/or wherein the first dose is administeredat a first lipid dose rate between 15 and 25 μg/kg/min, and wherein thesecond dose is administered at a second lipid dose rate between 55 and75 μg/kg/min.

In certain embodiments of the aforesaid methods, the first dose isadministered at a first siRNA dose rate between 1.5 and 2 μg/kg/min, andwherein the second dose is administered at a second siRNA dose ratebetween 4 and 6 μg/kg/min;

In other embodiments of the aforesaid methods, the first lipid dose rateis between 15 and 25 μg/kg/min, and the second lipid dose rate isbetween 55 and 75 μg/kg/min.

In other embodiments of the aforesaid methods, the first dose isadministered at between 0.5 and 1.5 mL/min, and the second dose isadministered at between 2.5 and 3.5 mL/min.

In other embodiments of the aforesaid methods, the first dose isadministered over a period of between 10 and 20 minutes, and the seconddose is administered over a period of between 50 and 60 minutes.

In other embodiments of the aforesaid methods, the first siRNA dose isbetween 20 and 30 μg/kg, and the second siRNA dose is between 250 and300 μg/kg.

In other embodiments of the aforesaid methods, the first lipid dose isbetween 250 and 400 μg/kg, and the second lipid dose is between 2500 and4000 μg/kg.

In other embodiments of the aforesaid methods, the total siRNA dose inthe first and the second doses are between 0.2 and 0.4 μg/kg, e.g., 0.3μg/kg.

In other embodiments of the aforesaid methods, the total lipid dose inthe first and the second doses are between 3.0 and 4.5 μg/kg

In other embodiments of the aforesaid methods, the first siRNA dose rateis between 0.1 and 0.15 mg/min, and the second siRNA dose rate isbetween 0.3 and 0.4 mg/min.

In other embodiments of the aforesaid methods, the first lipid dose rateis between 1.0 and 1.5 mg/min, and the second lipid dose rate is between3.5 and 4.5 mg/min.

In other embodiments of the aforesaid methods, the first siRNA dose isbetween 1.5 and 2.0 mg and the second siRNA dose is between 15 and 25mg.

In other embodiments of the aforesaid methods, the first lipid dose isbetween 15 and 25 mg and the second lipid dose is between 200 and 300mg.

In other embodiments of the aforesaid methods, the lipid formulation isan LNP11 formulation.

IRR, Hypersensitivity Reaction and Biomarker Detection

A hypersensitivity reaction may occur as the doses of the compositionsdescribed herein are increased (e.g., at a dose of about 300 μg/kg). Inthose embodiments of increased doses, the subject can be treated withthe methods and dosage regiments described herein. Alternatively, or incombination, the subject may be premedicated with one or more of asteroid, a histamine blocker or acetaminophen.

In some embodiments, the dosages and time periods of administration ofthe doses decribed herein are selected such that no substantial IRRand/or hypersensitivity reaction (e.g., a detectable hypersensitivityreaction) occurs in a subject. The IRR or the hypersensitivity reactioncan be an acute reaction (e.g., can occur during dose administration orcan start after the second dose administration is completed). In someembodiments, the subject is a human. Alternatively, the subject can bean animal (e.g., an animal model for a complement-mediatedhypersensitivity reaction (e.g., a porcine model as described herein)).In yet other embodiments, the subject is suffering from a disorderrelated to expression of one or more of the target genes disclosedherein, or is at risk of developing a disorder related to expression ofthe target gene.

In certain embodiments, the method described herein further include thestep of evaluating the subject after administration of the first dose,the second dose, or both, for the presence of one or more of thefollowing: a skin reaction (e.g., urticaria, erythema, edema, rash,pruritus, eruptions), a hemodynamic change, e.g., a change in bloodpressure (e.g., hypotension or hypertension), a respiratory problem(e.g., laryngospasm, laryngeal edema, bronchospasm, dyspnea), pain(e.g., joint pain, back pain, abdominal pain or chest pain), or othermanifestations of hypersensitivity (e.g., one or more of fever, chills,nausea, vomiting or neurological changes). In one embodiment, thedetection step includes evaluating one or more cardiovascularparameters.

Alternatively, or in combination, the methods described herein furtherinclude the step of evaluating the subject after administration of thefirst dose, the second dose, or both, for a change in a complementmarker, e.g., complement activation (e.g., a change in one or morecomplement factors chosen from Bb or C3a^(b)), wherein an increase thelevel of a complement biomarker is indicative of a hypersensitivityreaction.

A change in a complement marker can be detected in vivo or using an invitro assay. For example, a change in complement activation can bedetected using an assay that detects a complement cascade component,e.g, an assay (e.g., ELISA) that detects one or more of: totalcomplement proteins (e.g., C3 and C5); complement split products (e.g.,Bb, C3a or C5a), or terminal complement complement complex: sC5b-9.Additional examples of in vitro assays for evaluating complementactivity include CH50: Residual total hemolytic complement activity, orAH50: Residual alternative pathway of hemolytic complement activity.Alternatively, a change in complement activation can be detected usingan animal model. In embodiments, a sample to be evaluated can beobtained from a subject exposed to the compositions described herein.For example, the sample can be a serum/plasma sample obtained for an invivo assay. In other embodiments, naïve serum/plasma can be used formodeling complement activation in vitro.

Alternatively, or in combination, the methods described herein furtherinclude the step of evaluating the subject after administration of thefirst dose, the second dose, or both, for a change in thromboxanelevels, e.g., thromboxane B2 in plasma, e.g., wherein an increase in thelevel of thromboxane is indicative of an increased hypersensitivityreaction, e.g., an increased acute hypersensitivity reaction.

Alternatively, or in combination, the methods described herein furtherinclude the step of evaluating the subject after administration of thefirst dose, the second dose, or both, or changes in one or morecytokines chosen from interferon-alpha, interferon-gamma, tumor necrosisfactor-alpha, interleukin lbeta, interleukin 1 receptor antagonist(IL-1RA), interleukin-6, interleukin-8, interleukin-12, interleukin-18,interferon inducing protein-10, granulocyte colony stimulating factor,or C-reactive protein (CRP). In certain embodiments, an increase in thelevel of IL-6, IL-8, IL-1RA or CRP is indicative of an increasedhypersensitivity reaction, e.g., relative to a reference parameter(e.g., a subject exposed to a bolus dose, or the subject prior totreatment).

In some embodiments, the methods described herein do not cause adetectable hypersensitivity reaction, e.g., as measured by one or moreof the assays or symptoms described herein. In embodiments, the methodresults in a decrease in the hypersensitivity reaction, which is lessthan 1%, 5%, 10%, 25%, 30%, 35% or 40%, e.g., as measured by one or moreof of the assays or symptoms described herein. In certain embodiments,the changes described herein are compared to a reference parameter(e.g., a subject exposed to a bolus dose, or the subject prior totreatment).

In one embodiment, in response to the first and second dose regimendisclosed herein, the subject shows a reduced hypersensitivity reaction(e.g., a decreased hemodynamic change, relative to a reference parameter(e.g., a subject exposed to a bolus dose, or the subject prior totreatment).

In certain embodiments, the methods described herein cause a reducedhypersensitivity reaction, e.g., leading to a reduction (e.g., partialor complete reduction) in the administration of one or more of a steroid(e.g., dexamethasone or an equivalent), an analgesic (e.g.,paracetamol), or a histamine receptor antagonist (e.g., an H1 or an H2blocker). In other embodiments, the subject does not receiveadministration of a steroid (e.g., dexamethasone or an equivalent),within X hours of any of the initiation of administration of said firstdose, wherein X is less than 1 hour, 2 hours, 3 hours, 5 hours, 10hours, 15 hours, 24 hours or 48 hours.

Lipid Formulations

In some embodiments, the lipid formulation of the composition is alipid-nucleic acid particle, e.g., a nanoparticle. In one embodiment,the nucleic acid molecules described herein are formulated in a stablenucleic acid lipid particle (SNALP). In embodiments, the lipid-nucleicacid particle has a mean diameter of about 50 nm to about 200 nm, e.g.,about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm toabout 110 nm, or about 70 nm to about 90 nm. In yet other embodiments,the lipid-nucleic acid particle has a mean diameter of from about 70 toabout 200 nm, e.g., from about 70 to about 150 nm, from about 120 toabout 200 nm, or from about 90 to about 130 nm.

Typically, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to RNAmolecule ratio) in the composition is in the range of from about 1:1 toabout 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1,from about 4:1 to about 13:1, from about 4:1 to about 10:1, from about5:1 to about 9:1, from about 6:1 to about 9:1, or about 13:1, about12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about6:1, about 5:1. In certain embodiments, the lipid to drug ratio(mass/mass ratio) (e.g., lipid to RNA molecule ratio) is in the range offrom about 5:1 to 30:1, from about 6:1 to 25:1, or from about 10:1 to15:1. In yet other embodiments, the weight to weight ratio of thenucleic acid to the lipids in the lipid-nucleic acid particle is no lessthan about 0.1, e.g., greater than about 0.1, greater than about 0.2,greater than about 0.3, or greater than about 0.4, e.g., between about0.1 and about 0.4, or between about 0.2 and about 0.3. In anotherembodiment, the ratio of lipid:nucleic acid is at least about 1:1, atleast about 2:1, at least about 3:1, at least about 4:1, at least about5:1, at least about 6:1, at least about 7:1, at least about 8:1, atleast about 10:1, at least about 11:1, at least about 12:1, at leastabout 15:1, or at least 30:1, e.g., between about 0.5:1 to about 15:1,about 1:1 to about 20:1, about 3:1 to about 15:1, about 4:1 to about15:1, or about 5:1 to about 13:1.

The lipid can be a cationic or a non-cationic lipid, or a combinationthereof. In other embodiments, the lipid formulation comprises alipid-nucleic acid particle comprising a cationic lipid, a non-cationiclipid, a PEG-lipid conjugate. The lipid formulation can further includea sterol, e.g., a cholesterol.

In embodiments, the cationic lipid comprises from about 20 mol % toabout 60 mol %, or about 40 mol % of the total lipid present in theformulation. In other embodiments, the cationic lipid comprises fromabout 2% to about 55%, e.g., from about 5% to about 45%, from about 10%to about 40%, from about 5% to about 15%, or from about 40% to about50%, by weight of the total lipid present in the lipid formulation.Examples of cationic lipid include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), bis(3-pentyloctyl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate,1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. Other lipids are described, e.g., inU.S. Pat. Nos. 5,976,567, 6,858,225, and 6,825,432, which areincorporated herein by references. In one embodiment, the cationic lipidis selected from the group consisting of DODAC, DDAB, DOTAP, DOTMA,DOSPA, DMRIE, DOGS, DC-Chol, and combinations thereof.

In embodiments, the lipid of the formulation is a non-cationic lipid,e.g., an anionic lipid or a neutral lipid. In embodiments, thenon-cationic lipid comprises from about 5 mol % to about 90 mol %, about10 mol %, or about 58 mol % if cholesterol is included, of the totallipid present in the formulation. In other embodiments, the non-cationiclipid comprises from about 37% to about 89%, e.g., from about 37% toabout 75%, or from 40% to about 70%, by weight of the total lipidpresent in the lipid formulation. Examples of cationic lipid include,but are not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. In yet other embodiments, the non-cationic lipid isselected from the group consisting of DOPE, POPC, EPC, ESM, polyethyleneglycol-based polymers, and combinations thereof.

A lipid component of the formulation can be conjugated or modified,e.g., to prevent or reduce aggregation of particles. In certainembodiments, the conjugated lipid is from about 0 mol % to about 20 mol%, e.g., from about 1% to about 15%, or about 2 mol % of the total lipidpresent in the formulation.

In one embodiment, the conjugated or modified lipid is a polyethyleneglycol-modified lipid, e.g., a polyethylene glycol-modified ceramide, ora polyamide oligomer-modified lipid. In one embodiment, the PEG-lipidcomprises from about 1% to about 15%, e.g., from about 3% to about 12%,e.g., about 10%, by weight of the total lipid present in theformulation. In embodiments, the PEG or PEG-modified lipid is present inthe lipid formulation in a molar amount from about 0.5% to about 20%,e.g., about 0.5% to about 10%, about 0.5% to about 5%, about 1.5%, about0.5%, about 1.5%, about 3.5%, or about 5%. The PEG or PEG-modified lipidcan comprise a PEG molecule of an average molecular weight of no greaterthan 2,000 Da, e.g., about 2,000 Da, about 1,500 Da, about 1,000 Da, orabout 500 Da.

The PEG-conjugated lipid can be chosen from a polyethyleneglycol(PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), aPEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), ora mixture thereof. The PEG-DAA conjugate may be, for example, aPEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), aPEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (Ci₈). In oneembodiment, the modified lipid is PEG-CerC14 or PEG-CerC20. In yet otherembodiments, the lipid formulation further comprises a PEG orPEG-modified lipid (e.g., a PEG-modified lipid chosen from one or moreof PEG-C₁₄ to PEG-C₂₂, PEG-Cer₁₄ to PEG-C₂₀, or PEG-DSPE).

In embodiments, the lipid-nucleic acid particle further includes acholesterol at, e.g., about 10 mol % to about 60 mol %, about 15% toabout 50%, or about 48 mol % of the total lipid present in theformulation.

In some embodiments, the lipid formulation comprises a lipid-nucleicacid particle comprising a lipid layer surrounding and encapsulating acentral region containing a nucleic acid (e.g., a polyanionic nucleicacid), wherein the lipid layer comprises a titratable lipid comprising aprotonatable group having a pKa of from about 4 to about 11 (e.g., a pKais from about 4 to about 7, from about 4 to about 6, or from about 5 toabout 7). In one embodiment, the titratable lipid is an amino lipid,e.g., DODAP.

In one embodiment, the nucleic acid in the lipid formulation is notsubstantially degraded after incubation in serum at 37° C. for 30minutes.

In certain embodiments, the lipid formulation comprises DLinDMA, MC3 orC12-200. Exemplary lipid formulation can include a cationic lipid ofFormula I/MC3 (also called DLin-M-C3-DMA, MC3 or M-C3 described in,e.g., herein and in U.S. Pat. No. 8,158,601 incorporated by reference,and referred to herein as “MC3” or “Formula I/MC3”), or apharmaceutically acceptable salt thereof. In certain embodiments, thecationic lipid of Formula I/MC3 is present in the lipid formulation in amolar amount from about 25% to about 75%, e.g., from about 35% to about65%, from about 45% to about 65%, about 60%, about 57.5%, about 50%, orabout 40%. In one embodiment, the lipid formulation further comprises aneutral lipid (e.g., a neutral lipid chosen from DSPC, DPPC, DMPC, POPC,DOPE or SM, or a combination thereof. In embodiments, the neutral lipidis present in the lipid formulation in a molar amount from about 0.5% toabout 15%, e.g., from about 3% to about 12%, from about 5% to about 10%,about 15%, about 10%, or about 7.5%. In embodiments, the lipidformulation further comprises a sterol, e.g., a cholesterol. The sterolcan present in the lipid formulation in a molar amount from about 5% toabout 50%, e.g., about 15% to about 45%, about 20% to about 40%, about40%, about 38.5%, about 35%, or about 31%. In yet other embodiments, thelipid formulation further comprises a PEG or PEG-modified lipid (e.g., aPEG-modified lipid chosen from one or more of PEG-C₁₄ to PEG-C₂₂,PEG-Ceri4 to PEG-C₂₀, or PEG-DSPE).

In one embodiment, the lipid formulation comprises a cationic lipid ofFormula I/MC3, a neutral lipid, a sterol, and a PEG or PEG-modifiedlipid. In embodiments, the lipid formulations can comprise:

(i) about 25-75% of cationic lipid of Formula I/MC3, about 0.5-15% ofthe neutral lipid, about 5-50% of the sterol, and about 0.5-20% of thePEG or PEG-modified lipid on a molar basis;

(ii) about 35-65% of cationic lipid of Formula I/MC3, about 3-12% of theneutral lipid, about 15-45% of the sterol, and about 0.5-10% of the PEGor PEG-modified lipid on a molar basis;

(iii) about 45-65% of cationic lipid of Formula I/MC3, about 5-10% ofthe neutral lipid, about 25-40% of the sterol, and about 0.5-10% of thePEG or PEG-modified lipid on a molar basis;

(iv) about 40-65% of cationic lipid of Formula I/MC3, about 5-10% of aneutral lipid, about 25-40% of a sterol, and about 0.5-10% of a PEG orPEG-modified lipid; (v) about 50% of cationic lipid of Formula I, about10% of the neutral lipid (e.g., DSPC), about 38.5% of the sterol (e.g.,cholesterol), and about 1.5% of the PEG or PEG-modified lipid (e.g.,PEG-DMG);

(v) about 50% of cationic lipid of Formula I/MC3, about 10% of theneutral lipid, about 35% of the sterol, and about 5% of the PEG orPEG-modified lipid; or

(vi) about 57.2% of cationic lipid of Formula I/MC3, about 7.1% of theneutral lipid, about 34.3% of the sterol, and about 1.4% of the PEG orPEG-modified lipid.

In one embodiment, the lipid formulation comprises about 50% of cationiclipid of Formula I/MC3, about 10% of the neutral lipid (e.g., DSPC),about 38.5% of the sterol (e.g., cholesterol), and about 1.5% of the PEGor PEG-modified lipid (e.g., PEG-DMG). This lipid formulation is alsoreferred to herein as LNP-11 (see e.g., Table 1).

The formulations described herein can be prepared by an in-line mixingmethod. In other embodiments, the formulation is prepared by anextrusion method.

In yet other embodiments, the lipid formulation can further comprises atleast one apolipoprotein (e.g., an ApoE, active polymorphic forms,isoforms, variants and mutants, and fragments or truncated formsthereof).

In other embodiments, the lipid formulation further comprises atargeting lipid, e.g., N-acetyl galactosamine. The N-acetylgalactosamide can comprise at least a mono-, bi- or a triantennary sugarunit. In embodiments, the targeting lipid is present in the formulationin a molar amount of from about 0.001% to about 5%, e.g., from about0.005% to about 1.5%, e.g., about 0.005%, about 0.15%, about 0.3%, about0.5%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, or about 5%.

In other embodiments, the targeting lipid is a compound selected fromthe group consisting of GalNAc3-DSG (e.g., referred to as Formula II inU.S. Pat. No. 8,158,601), GalNAc3-PEG-DSG (e.g., referred to as FormulaIII in U.S. Pat. No. 8,158,601)), (GalNAc)₃-PEG-LCO (e.g., referred toas Formula IV in U.S. Pat. No. 8,158,601), Folate-PEG2000-DSG (e.g.,referred to as Formula VI in U.S. Pat. No. 8,158,601), andFolate-PEG3400-DSG (e.g., referred to as Formula VII in U.S. Pat. No.8,158,601).

Nucleic Acid/RNA Molecules

In some embodiments, the nucleic acid molecule in the composition ischosen from: double stranded RNA (dsRNA) molecules, single-stranded RNAimolecules, microRNA (miRNA), antisense RNA, short hairpin RNA (shRNA),antagomirs, mRNA, decoy RNA, DNA, plasmids or aptamers. In oneembodiment, the nucleic acid molecule is an RNA molecule, e.g., an RNAmolecule as described herein (e.g., an RNA molecule capable of mediatingRNA interference or an iRNA). In one embodiment, the RNA molecule isdouble-stranded. In embodiments, the RNA molecule comprises a sense andan antisense strand. For example, the RNA molecule is a dsRNA that formsa duplex structure between 15 and 30 basepairs in length. In oneembodiment, the region of complementarity between the strands is atleast 17 nucleotides in length (e.g., between 19 and 25, e.g., between19 and 21, nucleotides in length). In embodiments, each strand of thenucleic acid (e.g., RNA) molecule is no more than 30 nucleotides inlength.

In other embodiments, the nucleic acid (e.g., RNA) molecules included inthe compositions encompass a dsRNA having an RNA strand (the antisensestrand) having a region, e.g., a region that is 30 nucleotides or less,generally 19-24 nucleotides in length, that is substantiallycomplementary to at least part of a target mRNA.

In other embodiments, the nucleic acid (e.g., RNA) molecule is asingle-stranded molecule, e.g., comprises an antisense strand.

In some embodiments, the nucleic acid (e.g., RNA) molecule is 19-21nucleotides in length. In some embodiments, the iRNA is 19-21nucleotides in length and is in a lipid formulation, e.g. a lipidnanoparticle (LNP) formulation (e.g., an LNP11 formulation).

In other embodiments, the nucleic acid (e.g., RNA) molecule is 21-23nucleotides in length.

In some embodiments, the nucleic acid (e.g., RNA) molecule is from about15 to about 25 nucleotides in length, and in other embodiments thenucleic acid (e.g., RNA) molecule is from about 25 to about 30nucleotides in length. The nucleic acid (e.g., RNA) molecule can inhibitthe expression of a target gene by at least 10%, at least 20%, at least25%, at least 30%, at least 35% or at least 40% or more, such as whenassayed by a method as described herein.

In other embodiments, the nucleic acid (e.g., RNA) molecule comprises atleast one modified nucleotide. The modified nucleotides can be chosenfrom one or more of of: a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, and a terminal nucleotide linkedto a cholesteryl derivative or dodecanoic acid bisdecylamide group; or a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide.

In other embodiments, least one strand of the nucleic acid (e.g., RNA)molecule comprises a 3′ overhang of at least 2 nucleotides. In otherembodiments, one end of the double-stranded molecule is blunt-ended.

In embodiments, the nucleic acid (e.g., RNA) molecule has a sequencehaving an identity of at least 70 percent (e.g., 80%, 90%, 95% orhigher) to a target mRNA. In one embodiment, the nucleic acid (e.g.,RNA) molecule has a sequence complementary (e.g., is fully complementaryor substantially complementary) to a target mRNA.

In certain embodiments, the target mRNA is chosen from a mammalian,plant, pathogen-associated, viral, or disease-associated mRNA. Thetarget mRNA may be associated with a disease, e.g., a tumor-associatedmRNA, or an autoimmune disease-associated mRNA.

Target genes can be chosen from: Factor VII, Eg5, PCSK9, TPX2, apoB,SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21)gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, HAMP, Activated ProteinC gene, Cyclin D gene, VEGF gene, antithrombin 3 gene, aminolevulinatesynthase 1 gene, alpha-1-antitrypsin gene, tmprss6 gene, apoa1 gene,apoc3 gene, bc11a gene, klf gene, angpt13 gene, p1k gene, PKN3 gene,HBV, HCV, p53 gene, angiopoietin gene, angiopoietin-like 3 gene,complement component 3 (C3) gene, or complement component 5 (C5) gene.In certain embodiments, the target is chosen from: Eg5, PCSK9, TTR,HAMP, VEGF gene, antithrombin 3 gene, aminolevulinate synthase 1 gene,alpha-1-antitrypsin gene, or tmprss6 gene.

In certain embodiments, the nucleic acid molecules as described hereintarget a wildtype target RNA transcript variant, a mutant transcript, ora combination thereof. For example, the nucleic acid molecule can targeta polymorphic variant, such as a single nucleotide polymorphism (SNP),of the target gene. In another embodiment, the nucleic acid moleculetargets both a wildtype and a mutant target gene transcript. In otherembodiments, the nucleic acid molecule targets a non-coding region ofthe target RNA transcript, such as the 5′ or 3′ untranslated region of atranscript.

Kits

In another aspect, the invention features a kit for administration of afirst dose and a second dose of a composition (e.g., a first and asecond dose as described herein). The kit includes:

providing a composition, said composition comprising a lipid formulationand a nucleic acid (e.g., RNA) molecule, wherein said second amount isgreater than said first amount; and

instruction for administration, wherein the first dose is instructed tobe administered over a time period that is no more than 1/X, wherein Xis 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time period over which thesecond dose is administered; and

the rate of administration, e.g., in mg/min or mL/min, of said firstdose is no more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40 or 50, of the rate of administration of said second dose;

Devices

In another aspect, the invention features a device, e.g., a device forintravenous administration, comprising:

a reservoir, e.g., a bag, containing a composition (e.g., a compositionthat can be administered at a first and/or a second dose as describedherein), said composition comprising a lipid formulation and a nucleicacid (e.g., RNA) molecule as described herein;

a conduit (e.g., tubing);

a means (e.g., a valve) for adjusting a rate of administration of thecomposition; and (optionally) a needle;

wherein said conduit communicates said reservoir to the means (e.g.,valve) (and optionally, a second conduit that connects to the needle),and wherein:

a) the composition is administered at a first dose (e.g., a first doseas described herein) over a time period that is no more than 1/X,wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time period over whichthe second dose is administered; or

b) the rate of administration, e.g., in mg/min or mL/min, of said firstdose is no more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40 or 50, of the rate of administration of said second dose.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the hemodynamic changes (PAP, SAP and HR) after firstbolus dose of siRNA-LNP formulation in pig 1.

FIG. 2 depicts the hemodynamic changes (PAP, SAP and HR) after firstbolus dose of siRNA-LNP formulation in pig 3.

FIG. 3 depicts the hemodynamic changes (PAP, SAP and HR) in pig01infused at a dose of 0.5 μg/kg in a 50 mL volume over 60 minutes.

FIG. 4 depicts the hemodynamic changes (PAP, SAP and HR) in pig06infused at a dose of 0.5 μg/kg in a 50 mL volume in two steps: 1/10^(th)of total dose over 15 minutes followed by the remaining 9/10^(th) of thedose over additional 60 minutes.

FIG. 5 is a bar graph showing the percentages of doses associated withinfusion-related reactions (IRRs) in patients administered a total doseof 0.30 μg/kg siRNA-LNP formulation by IV infusion at different doserates/regimens. The total dose was based on the amount of siRNA in theformulation. For the 60-minute regimen, the infusion occurred at a rateof 3 ml/min for the full 60 minutes. For the 70-minute regimen, theinfusion occurred at a rate of 1 ml/min for the first 15 minutes and 3ml/min for the remainder of the time.

DETAILED DESCRIPTION OF THE INVENTION

Infusion-related reactions (IRRs) relate to any signs or symptomsexperienced by a patient during infusion of a pharmacological orbiological agent (e.g., a drug). These reactions can be acute, andtypically occur during the first hour(s) or day after drugadministration. Acute infusion-related reactions include a variety ofsigns and symptoms, including, but not limited to, neurologic (e.g.,dizziness, headache, weakness, syncope, seizure), psychiatric (e.g.,anxiety), cardiovascular (e.g., tachycardia, hypotension, arrhythmia,chest pain, ischemia or infarction, cardiac arrest), cutaneous (e.g.,flushing, erythema, pruritus, urticaria, angioedema, maculopapularrash), and gastrointestinal signs and symptoms. Manifestations of IRRscan vary, and can include drug hypersensitivity reactions (reviewede.g., by Kang, S. P. and Saif, M. S. (2007) The Journal of SupportiveOncology, Vol. 5 (9):451-457).

Hypersensitivity reactions can be classified into immune mediated andnon-immune mediated hypersensitivity reactions. Immune mediatedhypersensitivity reactions relate to specific, adaptive immune responsesgenerated against an antigen, e.g., a drug. Non-immune mediatedhypersensitivity reactions relate to drug responses initiated by somepharmacological action of the drug, which can involve immune systemcomponents. Non-immune mediated hypersensitivity reactions, alsoreferred to as pseudoallergic or anaphylactoid reactions, have clinicalmanifestations that are often indistinguishable from allergic reactions.These reactions are believed to be associated with complementactivation, as well as degranulation of mast cells and/or basophilsleading to histamine release and an anaphylactic-like reaction.Non-immune mediated hypersensitivity reactions may involve cytokinerelease syndrome, either directly via pharmacology or indirectly throughantibody dependent cellular cytotoxity (ADCC). Id.

Without wishing to be bound by theory, Applicants have discovered thatinfusion reactions to compositions that include a lipid formulation andan RNA molecule appear to be associated with an IRR or ahypersensitivity reaction. For example, dose ranges of 0.1 to 1.25 μg/kgof siRNAs in human subjects premedicated with steroid, H1/H2 blockersand acetaminophen have been shown to activate a hypersensitivityresponse in about 15% of patients. These dose ranges are typicallyadministered as a continuous, single dose for a predetermined timeinterval, e.g., 60 minutes. The incidence of the IRR or hypersensitivityresponse correlates with lipid dose rate (for example, having athreshold of approx. 13 mg/min). Such IRR or hypersensitivity responseshave been reduced by readministration of the dose. Modifiedlipid-nucleic acid particles (e.g., LNP-11 formulated particles) withimproved efficacy have resulted in a reduction of the dose range to0.01-0.5 μg/kg siRNA, provided during an infusion interval over 60 mins.Subjects receiving the modified lipid-nucleic acid particles had a lipiddose rate associated with the hypersensitivity reaction of approx. 8mg/min. One subject showed a hypersensitivity response at higher doses(e.g., 0.5 μg/kg) to which the subject was able to complete the dose byslowing the infusion rate.

In one embodiment, it has been discovered that administration of a firstdose (or a pre-dose) of of a lipid-formulated RNA molecule correspondingto a portion of a second dose or the total dose, or administration of afirst dose at a portion of the rate of infusion of a second dose, over apre-treatment interval was found to reduce or prevent the IRR orhypersensitivity response in a subject. Accordingly, methods, kits anddevices for dosing a subject to reduce an IRR and/or a hypersensitivyresponse to a lipid-formulated RNA molecule are disclosed.

The term, “infusion-related reaction” or “TRW” relates to any sign orsymptom experienced by a subject, e.g., a patient, during infusion of apharmacological or biological agent (e.g., a drug). These reactions canbe acute, and typically occur during the first hours or a day after drugadministration. IRRs can include, but are not limited to, thehypersensitivity reactions described herein.

As used herein, the term “hypersensitivity reaction” encompass anyadverse event (e.g., immune-mediated and non-immune mediated) related toadministration of a pharmacological agent (e.g., an RNA molecule or aniRNA as described herein), regardless of etiology. In one embodiment,the hypersensitivity reaction is a non-immune mediated hypersensitivityreaction or a pseudoallergic reaction. Symptoms associated withhypersensitivity reactions (both immune and non-immune reactions)include, but are not limited to, flushing, various types of skin rashes(e.g., urticaria, erythema, edema, rash, pruritus, eruptions);hemodynamic changes, e.g., a change in blood pressure (e.g., hypotensionor hypertension) and/or heart rate; respiratory problems (e.g.,laryngospasm, laryngeal edema, bronchospasm, dyspnea, or chestdiscomfort); pain (e.g., joint pain, back pain, abdominal pain or chestpain); or other manifestations of hypersensitivity (e.g., one or more offever, chills, nausea, gastrointestinal disturbances (e.g., vomiting),hypoxia, neurological changes, or cardiac arrest or shock.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent”refers to an agent that contains RNA as that term is defined herein, andwhich mediates the targeted cleavage of an RNA transcript, e.g., via anRNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNAas described herein effects inhibition of TTR expression. Inhibition oftarget gene expression may be assessed based on a reduction in the levelof target gene mRNA or a reduction in the level of the target geneprotein. As used herein, “target sequence” refers to a contiguousportion of the nucleotide sequence of an mRNA molecule formed during thetranscription of a target gene, including mRNA that is a product of RNAprocessing of a primary transcription product. The target portion of thesequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion. For example, the targetsequence will generally be from 9-36 nucleotides in length, e.g., 15-30nucleotides in length, including all sub-ranges therebetween. Asnon-limiting examples, the target sequence can be from 15-30nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides,15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides,18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides,19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24nucleotides,20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides,21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they may form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding a target gene protein). For example, apolynucleotide is complementary to at least a part of a target gene mRNAif the sequence is substantially complementary to a non-interruptedportion of an mRNA encoding target gene. As another example, apolynucleotide is complementary to at least a part of a target gene mRNAif the sequence is substantially complementary to a non-interruptedportion of an mRNA encoding target gene.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to aniRNA that includes an RNA molecule or complex of molecules having ahybridized duplex region that comprises two anti-parallel andsubstantially complementary nucleic acid strands, which will be referredto as having “sense” and “antisense” orientations with respect to atarget RNA. The duplex region can be of any length that permits specificdegradation of a desired target RNA, e.g., through a RISC pathway, butwill typically range from 9 to 36 base pairs in length, e.g., 15-30 basepairs in length. Considering a duplex between 9 and 36 base pairs, theduplex can be any length in this range, for example, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, or 36 and any sub-range therein between, including, butnot limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs,15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs,15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs,18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs,19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs,19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs,20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs,20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs,21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAsgenerated in the cell by processing with Dicer and similar enzymes aregenerally in the range of 19-22 base pairs in length. One strand of theduplex region of a dsDNA comprises a sequence that is substantiallycomplementary to a region of a target RNA. The two strands forming theduplex structure can be from a single RNA molecule having at least oneself-complementary region, or can be formed from two or more separateRNA molecules. Where the duplex region is formed from two strands of asingle molecule, the molecule can have a duplex region separated by asingle stranded chain of nucleotides (herein referred to as a “hairpinloop”) between the 3′-end of one strand and the 5′-end of the respectiveother strand forming the duplex structure. The hairpin loop can compriseat least one unpaired nucleotide; in some embodiments the hairpin loopcan comprise at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 20, at least 23 or moreunpaired nucleotides. Where the two substantially complementary strandsof a dsRNA are comprised by separate RNA molecules, those molecules neednot, but can be covalently connected. Where the two strands areconnected covalently by means other than a hairpin loop, the connectingstructure is referred to as a “linker.” The term “siRNA” is also usedherein to refer to a dsRNA as described above.

In another embodiment, the iRNA agent may be a “single-stranded siRNA”that is introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In another aspect, the RNA agent is a “single-stranded antisense RNAmolecule”. An single-stranded antisense RNA molecule is complementary toa sequence within the target mRNA. Single-stranded antisense RNAmolecules can inhibit translation in a stoichiometric manner by basepairing to the mRNA and physically obstructing the translationmachinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355.Alternatively, the single-stranded antisense molecules inhibit a targetmRNA by hydridizing to the target and cleaving the target through anRNaseH cleavage event. The single-stranded antisense RNA molecule may beabout 10 to about 30 nucleotides in length and have a sequence that iscomplementary to a target sequence. For example, the single-strandedantisense RNA molecule may comprise a sequence that is at least about10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from any one of the antisense nucleotide sequences describedherein.

The term “nucleic acid molecule” encompasses an RNA molecule (e.g., anRNA molecule as described herein), a DNA molecule (e.g., a 100%deoxynucleoside-containing molecule), and a combination of an RNA and aDNA molecule. It includes a naturally-occurring andnon-naturally-occurring nucleic acid molecule. In one embodiment, thenucleic acid molecule is isolated or purified. In one embodiment, thenucleic acid molecule is synthetic (e.g., chemically synthesized) orrecombinant. In other embodiments, the nucleic acid molecule is anon-naturally-occurring nucleic acid molecule, e.g., an analog or aderivative of a nucleic acid molecule, e.g., analogs and derivatives ofDNA, RNA or both. For example, the nucleic acid molecule can include oneor more nucleotide/nucleoside analogs or derivatives as described hereinor as known in the art. In certain embodiments, “nucleic acid molecule”includes an oligonucleotide molecule (e.g., a single-stranded or adouble-stranded oligonucleotide (e.g., an oligodeoxyribo- or anoligoribonucleotide, or a combination thereof)). In other embodiments,“nucleic acid molecule” includes an RNA molecule, e.g., asingle-stranded or a double-stranded RNA (dsRNA), e.g., as describedherein. In certain embodiments, the nucleic acid molecule comprises atleast 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95% or 100% deoxyribonucleosides, e.g., in one or both strands.

The term “RNA molecule” or “ribonucleic acid molecule” encompasses anaturally-occurring and non-naturally-occurring RNA molecule. In oneembodiment, the RNA molecule is isolated or purified. In one embodiment,the RNA molecule is synthetic (e.g., chemically synthesized) orrecombinant. In other embodiments, the RNA molecule is anon-naturally-occurring RNA molecule, e.g., an analog or a derivative ofan RNA molecule. In certain embodiments, the RNA molecule comprises oneor more ribonucleotide/ribonucleoside analogs or derivatives asdescribed herein or as known in the art. A “ribonucleoside” includes anucleoside base and a ribose sugar, and a “ribonucleotide” is aribonucleoside with one, two or three phosphate moieties. However, theterms “ribonucleoside” and “ribonucleotide” can be considered to beequivalent as used herein. The ribonucleoside or ribonucleotide can bemodified in the nucleobase structure or in the ribose-phosphate backbonestructure, e.g., as described herein below. In certain embodiments, theRNA molecule that comprises a ribonucleoside analog or derivativeretains the ability to form a duplex. As non-limiting examples, an RNAmolecule can also include at least one modified ribonucleoside,including but not limited to, a 2′-O-methyl modified nucleoside, anucleoside comprising a 5′ phosphorothioate group, a terminal nucleosidelinked to a cholesteryl derivative or dodecanoic acid bisdecylamidegroup, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoromodified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modifiednucleoside, morpholino nucleoside, a phosphoramidate or a non-naturalbase comprising nucleoside, or any combination thereof. Alternatively,an RNA molecule can comprise at least two modified ribonucleosides, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20 or more, up to the entirelength of the RNA molecule. The modifications need not be the same foreach of such a plurality of modified ribonucleosides in an RNA molecule.In one embodiment, modified RNA molecules contemplated for use inmethods and compositions described herein include peptide nucleic acids(PNAs) that have the ability to form the required duplex structure andthat permit or mediate the specific degradation of a target RNA, e.g.,via a RISC pathway.

Exemplary RNA molecules, include but are not limited to, iRNA agents ormolecules, double stranded RNA (dsRNA) molecules, siRNA molecules,single-stranded RNAi molecules, single-stranded siRNA molecules,microRNA (miRNA), antisense RNA, short hairpin RNA (shRNA), antagomirs,mRNA, decoy RNA, vectors and aptamers.

In certain embodiments, an RNA molecule comprises a deoxyribonucleoside.For example, the RNA molecule, e.g., an iRNA agent, can comprise one ormore deoxynucleosides, including, for example, a deoxynucleosideoverhang(s), or one or more deoxynucleosides within the double strandedportion of a dsRNA. In certain embodiments, the RNA molecule comprises apercentage of deoxyribonucleoses of at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or higher (but not 100%)deoxyribonucleosides, e.g., in one or both strands. In certainembodiments, the term “iRNA” does not encompass a double stranded DNAmolecule (e.g., a naturally-occurring double stranded DNA molecule or a100% deoxynucleoside-containing DNA molecule).

In one aspect, an RNA interference agent includes a single stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleaves the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA that promotes the formationof a RISC complex to effect silencing of the target gene.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) may be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′ end, 3′ end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotideoverhang at the 3′ end and/or the 5′ end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/orthe 5′ end. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that issubstantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches may be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA or a plasmidfrom which an iRNA is transcribed. SNALPs are described, e.g., in U.S.Patent Application Publication Nos. 20060240093, 20070135372, and inInternational Application No. WO 2009082817. These applications areincorporated herein by reference in their entirety.

“Introducing into a cell,” when referring to an iRNA, means facilitatingor effecting uptake or absorption into the cell, as is understood bythose skilled in the art. Absorption or uptake of an iRNA can occurthrough unaided diffusive or active cellular processes, or by auxiliaryagents or devices. The meaning of this term is not limited to cells invitro; an iRNA may also be “introduced into a cell,” wherein the cell ispart of a living organism. In such an instance, introduction into thecell will include the delivery to the organism. For example, for in vivodelivery, iRNA can be injected into a tissue site or administeredsystemically. In vivo delivery can also be by a β-glucan deliverysystem, such as those described in U.S. Pat. Nos. 5,032,401 and5,607,677, and U.S. Publication No. 2005/0281781, which are herebyincorporated by reference in their entirety. In vitro introduction intoa cell includes methods known in the art such as electroporation andlipofection. Further approaches are described herein below or known inthe art.

As used herein, the term “modulate the expression of,” refers to at anleast partial “inhibition” or partial “activation” of a target geneexpression in a cell treated with an iRNA composition as describedherein compared to the expression of target gene in a control cell. Acontrol cell includes an untreated cell, or a cell treated with anon-targeting control iRNA.

The terms “activate,” “enhance,” “up-regulate the expression of,”“increase the expression of,” and the like, in so far as they refer to atarget gene, herein refer to the at least partial activation of theexpression of a target gene, as manifested by an increase in the amountof target gene mRNA, which may be isolated from or detected in a firstcell or group of cells in which a target gene is transcribed and whichhas or have been treated such that the expression of a target gene isincreased, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells).

In one embodiment, expression of a target gene is activated by at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administrationof an iRNA as described herein. In some embodiments, a target gene isactivated by at least about 60%, 70%, or 80% by administration of aniRNA featured in the invention. In some embodiments, expression of atarget gene is activated by at least about 85%, 90%, or 95% or more byadministration of an iRNA as described herein. In some embodiments, thetarget gene expression is increased by at least 1-fold, at least 2-fold,at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold,at least 500-fold, at least 1000 fold or more in cells treated with aniRNA as described herein compared to the expression in an untreatedcell. Activation of expression by small dsRNAs is described, forexample, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42,and in US20070111963 and US2005226848, each of which is incorporatedherein by reference.

The terms “silence,” “inhibit expression of,” “down-regulate expressionof,” “suppress expression of,” and the like, in so far as they refer toa target gene, herein refer to the at least partial suppression of theexpression of a target gene, as assessed, e.g., based on on target genemRNA expression, target gene protein expression, or another parameterfunctionally linked to target gene expression. For example, inhibitionof target gene expression may be manifested by a reduction of the amountof target gene mRNA which may be isolated from or detected in a firstcell or group of cells in which a target gene is transcribed and whichhas or have been treated such that the expression of a target gene isinhibited, as compared to a control. The control may be a second cell orgroup of cells substantially identical to the first cell or group ofcells, except that the second cell or group of cells have not been sotreated (control cells). The degree of inhibition is usually expressedas a percentage of a control level, e.g.,

(mRNA in control cells)−(mRNA in treated cells)/(mRNA in controlcells)·100%

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to target geneexpression, e.g., the amount of protein encoded by a target gene. Thereduction of a parameter functionally linked to target gene expressionmay similarly be expressed as a percentage of a control level. Inprinciple, target gene silencing may be determined in any cellexpressing target gene, either constitutively or by genomic engineering,and by any appropriate assay. However, when a reference is needed inorder to determine whether a given iRNA inhibits the expression of thetarget gene by a certain degree and therefore is encompassed by theinstant invention, the assays provided in the Examples below shall serveas such reference.

For example, in certain instances, expression of a target gene issuppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50% by administration of an iRNA featured in the invention. In someembodiments, a target gene is suppressed by at least about 60%, 65%,70%, 75%, or 80% by administration of an iRNA featured in the invention.In some embodiments, a target gene is suppressed by at least about 85%,90%, 95%, 98%, 99%, or more by administration of an iRNA as describedherein.

As used herein in the context of target gene expression, the terms“treat,” “treating,” “treatment,” and the like, refer to relief from oralleviation of pathological processes related to target gene expression.In the context of the present invention insofar as it relates to any ofthe other conditions recited herein below (other than pathologicalprocesses related to target gene expression), the terms “treat,”“treatment,” and the like mean to prevent, relieve or alleviate at leastone symptom associated with such condition, or to slow or reverse theprogression or anticipated progression of such condition. Thus, unlessthe context clearly indicates otherwise, the terms “treat,” “treatment,”and the like are intended to encompass prophylaxis, e.g., prevention ofdisorders and/or symptoms of disorders related to target geneexpression.

By “lower” in the context of a disease marker or symptom is meant astatistically or clinically significant decrease in such level. Thedecrease can be, for example, at least 10%, at least 20%, at least 30%,at least 40% or more, and is typically down to a level accepted aswithin the range of normal for an individual without such disorder.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes related to target geneexpression. The specificamount that is therapeutically effective can be readily determined by anordinary medical practitioner, and may vary depending on factors knownin the art, such as, for example, the type of pathological process, thepatient's history and age, the stage of pathological process, and theadministration of other agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of an iRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an iRNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, in amethod of treating a disorder related to target gene expression, aneffective amount includes an amount effective to reduce one or moresymptoms associated with the disease, or an amount effective to reducethe risk of developing conditions associated with the disease. Forexample, if a given clinical treatment is considered effective whenthere is at least a 10% reduction in a measurable parameter associatedwith a disease or disorder, a therapeutically effective amount of a drugfor the treatment of that disease or disorder is the amount necessary toeffect at least a 10% reduction in that parameter. For example, atherapeutically effective amount of an iRNA targeting target gene canreduce target gene protein levels by any measurable amount, e.g., by atleast 10%, 20%, 30%, 40% or 50%.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. Agents included in drug formulations aredescribed further herein below.

The term “about” when referring to a number or a numerical range meansthat the number or numerical range referred to is an approximationwithin experimental variability (or within statistical experimentalerror), and thus the number or numerical range may vary from, forexample, between 1% and 15% of the stated number or numerical range.

Double-Stranded Ribonucleic Acid (dsRNA)

Described herein are iRNA agents that inhibit the expression of a targetgenegene. In one embodiment, the iRNA agent includes double-strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of atarget gene in a cell or in a subject (e.g., in a mammal, e.g., in ahuman), where the dsRNA includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of a target genegene, and where the region ofcomplementarity is 30 nucleotides or less in length, generally 19-24nucleotides in length, and where the dsRNA, upon contact with a cellexpressing the target gene, inhibits the expression of the target geneby at least 10% as assayed by, for example, a PCR or branched DNA(bDNA)-based method, or by a protein-based method, such as by Westernblot. In one embodiment, the iRNA agent activates the expression of atarget gene in a cell or mammal. Expression of a target gene in cellculture, such as in COS cells, HeLa cells, primary hepatocytes, HepG2cells, primary cultured cells or in a biological sample from a subjectcan be assayed by measuring target gene mRNA levels, such as by bDNA orTaqMan assay, or by measuring protein levels, such as byimmunofluorescence analysis, using, for example, Western Blotting orflow cytometric techniques.

A dsRNA includes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure under conditions in which the dsRNAwill be used. One strand of a dsRNA (the antisense strand) includes aregion of complementarity that is substantially complementary, andgenerally fully complementary, to a target sequence, derived from thesequence of an mRNA formed during the expression of a target gene. Theother strand (the sense strand) includes a region that is complementaryto the antisense strand, such that the two strands hybridize and form aduplex structure when combined under suitable conditions. Generally, theduplex structure is between 15 and 30 inclusive, more generally between18 and 25 inclusive, yet more generally between 19 and 24 inclusive, andmost generally between 19 and 21 base pairs in length, inclusive.Similarly, the region of complementarity to the target sequence isbetween 15 and 30 inclusive, more generally between 18 and 25 inclusive,yet more generally between 19 and 24 inclusive, and most generallybetween 19 and 21 nucleotides in length, inclusive. In some embodiments,the dsRNA is between 15 and 20 nucleotides in length, inclusive, and inother embodiments, the dsRNA is between 25 and 30 nucleotides in length,inclusive. As the ordinarily skilled person will recognize, the targetedregion of an RNA targeted for cleavage will most often be part of alarger RNA molecule, often an mRNA molecule. Where relevant, a “part” ofan mRNA target is a contiguous sequence of an mRNA target of sufficientlength to be a substrate for RNAi-directed cleavage (i.e., cleavagethrough a RISC pathway). dsRNAs having duplexes as short as 9 base pairscan, under some circumstances, mediate RNAi-directed RNA cleavage. Mostoften a target will be at least 15 nucleotides in length, e.g., 15-30nucleotides in length.

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of 9 to 36,e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that itbecomes processed to a functional duplex of e.g., 15-30 base pairs thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, then, a miRNA is a dsRNA. In another embodiment, a dsRNA isnot a naturally occurring miRNA. In another embodiment, an iRNA agentuseful to target gene expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein may further include one or moresingle-stranded nucleotide overhangs. The dsRNA can be synthesized bystandard methods known in the art as further discussed below, e.g., byuse of an automated DNA synthesizer, such as are commercially availablefrom, for example, Biosearch, Applied Biosystems, Inc. In oneembodiment, a target gene is a huma target gene. In another embodimentthe target gene is a mouse or a rat target gene. In specificembodiments, the first sequence is a sense strand of a dsRNA thatincludes a sense sequence, and the second sequence is an antisensestrand of a dsRNA that includes an antisense sequence. Alternative dsRNAagents that target sequences other than those of the dsRNAs disclosedherein can readily be determined using the target sequence and theflanking target gene sequence.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can be effective as well. In theembodiments described above, dsRNAs described herein can include atleast one strand of a length of minimally 21 nucleotides. It can bereasonably expected that shorter duplexes minus only a few nucleotideson one or both ends may be similarly effective as compared to the dsRNAsdescribed above. Hence, dsRNAs having a partial sequence of at least 15,16, 17, 18, 19, 20, or more contiguous nucleotides, and differing intheir ability to inhibit the expression of a target genegene by not morethan 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising thefull sequence, are contemplated according to the invention.

In addition, the RNAs identify a site in a target gene transcript thatis susceptible to RISC-mediated cleavage. As such, the present inventionfurther features iRNAs that target within one of such sequences. As usedherein, an iRNA is said to target within a particular site of an RNAtranscript if the iRNA promotes cleavage of the transcript anywherewithin that particular site. Such an iRNA will generally include atleast 15 contiguous nucleotides coupled to additional nucleotidesequences taken from the region contiguous to the selected sequence in atarget gene.

While a target sequence is generally 15-30 nucleotides in length, thereis wide variation in the suitability of particular sequences in thisrange for directing cleavage of any given target RNA. Various softwarepackages and the guidelines set out herein provide guidance for theidentification of optimal target sequences for any given gene target,but an empirical approach can also be taken in which a “window” or“mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that mayserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,it is contemplated that further optimization of inhibition efficiencycan be achieved by progressively “walking the window” one nucleotideupstream or downstream of the given sequences to identify sequences withequal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified furtheroptimization can be achieved by systematically either adding or removingnucleotides to generate longer or shorter sequences and testing thoseand sequences generated by walking a window of the longer or shortersize up or down the target RNA from that point. Again, coupling thisapproach to generating new candidate targets with testing foreffectiveness of iRNAs based on those target sequences in an inhibitionassay as known in the art or as described herein can lead to furtherimprovements in the efficiency of inhibition. Further still, suchoptimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart and/or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes, etc.) as an expressioninhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch not be located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent RNA strandwhich is complementary to a region of a target gene, the RNA strandgenerally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether an iRNA containing a mismatch to atarget sequence is effective in inhibiting the expression of a targetgene. Consideration of the efficacy of iRNAs with mismatches ininhibiting expression of a target gene is important, especially if theparticular region of complementarity in a target gene is known to havepolymorphic sequence variation within the population.

In one embodiment, at least one end of a dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties relative to their blunt-ended counterparts. In yetanother embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemicallymodified to enhance stability or other beneficial characteristics. Thenucleic acids featured in the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,(a) end modifications, e.g., 5′ end modifications (phosphorylation,conjugation, inverted linkages, etc.) 3′ end modifications (conjugation,DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases, (c) sugar modifications(e.g., at the 2′ position or 4′ position) or replacement of the sugar,as well as (d) backbone modifications, including modification orreplacement of the phosphodiester linkages. Specific examples of RNAcompounds useful in this invention include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkagesRNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In particular embodiments,the modified RNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, each of which is herein incorporated by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference.

Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′ position: OH; F; 0-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m) CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,C₁, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N3, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chin. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F) Similar modifications may alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

An iRNA may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, each of which is herein incorporated byreference in its entirety.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

iRNA Motifs

In one embodiment, the sense strand sequence may be represented byformula (I):

5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z ZZ)_(j)—N_(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7,8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(5t) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Ib);

5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′  (Ic); or

5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′n_(q)′-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and 1 is 0, or k is 0 and 1 is 1, or both kand 1 are 1.

The antisense strand can therefore be represented by the followingformulas:

5′n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p)′ 3′  (IIb);

5′n_(q)′-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p)′ 3′  (IIc); or

5′n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p),3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a)′ independently represents anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the antisense strand is represented as formula (ITC), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a)′ independently represents anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:

5′n_(p)′-N_(a)′-Y′Y′Y′-N_(a′)-n_(q),3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. Forexample, each nucleotide of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′,Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′n_(p)-N_(a)-(X X X)_(i)—N_(b)-Y Y Y-N_(b)-(Z ZZ)_(j)—N_(a)-n_(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not bepresent, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and 1 are 0; or both k and 1 are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

5′n_(p)-N_(a)-Y Y Y-N_(a)-n_(q)3′

3′n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′5′  (IIIa)

5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′

3′n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q)′5′  (IIIb)

5′n_(p)-N_(a)-X X X-N_(b)-YYY-N_(a)-n_(q)3′

3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′5′  (IIIc)

5′n_(p)-N_(a)-XXX-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q)3′

3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′5′  (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a), N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) andN_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker. In another embodiment, when the RNAi agent isrepresented by formula (IIId), the N_(a) modifications are 2′-O-methylor 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linkedto a neighboring nucleotide via phosphorothioate linkage, the sensestrand comprises at least one phosphorothioate linkage, and the sensestrand is conjugated to one or more GalNAc derivatives attached througha bivalent or trivalent branched linker

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes ca target the same gene or twodifferent genes; or each of the duplexes ca target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes ca target the same geneor two different genes; or each of the duplexes ca target same gene attwo different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to a ligand.Each of the agents ca target the same gene or two different genes; oreach of the agents ca target same gene at two different target sites.

iRNA Conjugates

The iRNA agents disclosed herein can be in the form of conjugates. Theconjugate may be attached at any suitable location in the iRNA molecule,e.g., at the 3′ end or the 5′ end of the sense or the antisense strand.The conjugates are optionally attached via a linker

In some embodiments, an iRNA agent described herein is chemically linkedto one or more ligands, moieties or conjugates, which may conferfunctionality, e.g., by affecting (e.g., enhancing) the activity,cellular distribution or cellular uptake of the iRNA. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994,4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med.Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In someembodiments, a ligand provides an enhanced affinity for a selectedtarget, e.g, molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Typical ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an a helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic.

In some embodiments, the ligand is a GalNAc ligand that comprises one ormore N-acetylgalactosamine (GalNAc) derivatives. Additional descriptionof GalNAc ligands is provided in the section titled CarbohydrateConjugates.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates,Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

Lipid Conjugates

In one embodiment, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule can typically bind a serum protein, suchas human serum albumin (HSA). An HSA binding ligand allows fordistribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA. A lipid based ligand canbe used to modulate, e.g., control (e.g., inhibit) the binding of theconjugate to a target tissue. For example, a lipid or lipid-based ligandthat binds to HSA more strongly will be less likely to be targeted tothe kidney and therefore less likely to be cleared from the body. Alipid or lipid-based ligand that binds to HSA less strongly can be usedto target the conjugate to the kidney.

In one embodiment, the lipid based ligand binds HSA. For example, theligand can bind HSA with a sufficient affinity such that distribution ofthe conjugate to a non-kidney tissue is enhanced. However, the affinityis typically not so strong that the HSA-ligand binding cannot bereversed.

In another embodiment, the lipid based ligand binds HSA weakly or not atall, such that distribution of the conjugate to the kidney is enhanced.Other moieties that target to kidney cells can also be used in place ofor in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as ahelical cell-permeation agent. In one embodiment, the agent isamphipathic. An exemplary agent is a peptide such as tat orantennopedia. If the agent is a peptide, it can be modified, including apeptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages,and use of D-amino acids. The helical agent is typically an α-helicalagent, and can have a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 1). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 2)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 3)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 4))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit is a cell targeting peptidesuch as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

An RGD peptide moiety can be used to target a particular cell type,e.g., a tumor cell, such as an endothelial tumor cell or a breast cancertumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGDpeptide can facilitate targeting of an dsRNA agent to tumors of avariety of other tissues, including the lung, kidney, spleen, or liver(Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGDpeptide will facilitate targeting of an iRNA agent to the kidney. TheRGD peptide can be linear or cyclic, and can be modified, e.g.,glycosylated or methylated to facilitate targeting to specific tissues.For example, a glycosylated RGD peptide can deliver an iRNA agent to atumor cell expressing avB3 (Haubner et al., Jour. Nucl. Med.,42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate comprises a monosaccharide.In one embodiment, the monosaccharide is an N-acetylgalactosamine(GalNAc). GalNAc conjugates are described, for example, in U.S. Pat. No.8,106,022, the entire content of which is hereby incorporated herein byreference. In some embodiments, the GalNAc conjugate serves as a ligandthat targets the iRNA to particular cells. In some embodiments, theGalNAc conjugate targets the iRNA to liver cells, e.g., by serving as aligand for the asialoglycoprotein receptor of liver cells (e.g.,hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or moreGalNAc derivatives. The GalNAc derivatives may be attached via a linker,e.g., a bivalent or trivalent branched linker. In some embodiments theGalNAc conjugate is conjugated to the 3′ end of the sense strand. Insome embodiments, the GalNAc conjugate is conjugated to the iRNA agent(e.g., to the 3′ end of the sense strand) via a linker, e.g., a linkeras described herein.

In some embodiments, the GalNAc conjugate is

In some embodiments, the RNAi agent is conjugated to L96 as defined inTable 2 and shown below

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker

Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXI)-(XXXIV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(p2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of 0, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), CC or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent,

NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO,CH═N-0,

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; andRa is H or aminoacid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)-O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Peptide-Based Cleavable Linking Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of which isherein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds, or “chimeras,” in the context of the presentinvention, are iRNA compounds, e.g., dsRNAs, that contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm, 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

Delivery of Nucleic Acid Molecules (iRNA)

The delivery of an iRNA to a subject in need thereof can be achieved ina number of different ways. In vivo delivery can be performed directlyby administering a composition comprising an iRNA, e.g. a dsRNA, to asubject. Alternatively, delivery can be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can beadapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992)Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporatedherein by reference in their entireties). However, there are threefactors that are important to consider in order to successfully deliveran iRNA molecule in vivo: (a) biological stability of the deliveredmolecule, (2) preventing non-specific effects, and (3) accumulation ofthe delivered molecule in the target tissue. The non-specific effects ofan iRNA can be minimized by local administration, for example by directinjection or implantation into a tissue (as a non-limiting example, atumor) or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that may otherwise beharmed by the agent or that may degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo.

Modification of the RNA or the pharmaceutical carrier can also permittargeting of the iRNA composition to the target tissue and avoidundesirable off-target effects. iRNA molecules can be modified bychemical conjugation to other groups, e.g., a lipid or carbohydrategroup as described herein. Such conjugates can be used to target iRNA toparticular cells, e.g., liver cells, e.g., hepatocytes. For example,GalNAc conjugates or lipid (e.g., LNP) formulations can be used totarget iRNA to particular cells, e.g., liver cells, e.g., hepatocytes.

Lipophilic groups such as cholesterol to enhance cellular uptake andprevent degradation. For example, an iRNA directed against ApoBconjugated to a lipophilic cholesterol moiety was injected systemicallyinto mice and resulted in knockdown of apoB mRNA in both the liver andjejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation ofan iRNA to an aptamer has been shown to inhibit tumor growth and mediatetumor regression in a mouse model of prostate cancer (McNamara, J O., etal (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment,the iRNA can be delivered using drug delivery systems such as ananoparticle, a dendrimer, a polymer, liposomes, or a cationic deliverysystem. Positively charged cationic delivery systems facilitate bindingof an iRNA molecule (negatively charged) and also enhance interactionsat the negatively charged cell membrane to permit efficient uptake of aniRNA by the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

Vector Encoded iRNAs

In another aspect, iRNA targeting the target gene can be expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299).Expression can be transient (on the order of hours to weeks) orsustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as aninverted repeat joined by a linker polynucleotide sequence such that thedsRNA has a stem and loop structure.

An iRNA expression vector is typically a DNA plasmid or viral vector. Anexpression vector compatible with eukaryotic cells, e.g., withvertebrate cells, can be used to produce recombinant constructs for theexpression of an iRNA as described herein. Eukaryotic cell expressionvectors are well known in the art and are available from a number ofcommercial sources. Typically, such vectors contain convenientrestriction sites for insertion of the desired nucleic acid segment.Delivery of iRNA expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from the patient followed by reintroduction into thepatient, or by any other means that allows for introduction into adesired target cell.

An iRNA expression plasmid can be transfected into a target cell as acomplex with a cationic lipid carrier (e.g., Oligofectamine) or anon-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e)SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors;(h) picornavirus vectors; (i) pox virus vectors such as an orthopox,e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and(j) a helper-dependent or gutless adenovirus. Replication-defectiveviruses can also be advantageous. Different vectors will or will notbecome incorporated into the cells' genome. The constructs can includeviral sequences for transfection, if desired. Alternatively, theconstruct may be incorporated into vectors capable of episomalreplication, e.g EPV and EBV vectors. Constructs for the recombinantexpression of an iRNA will generally require regulatory elements, e.g.,promoters, enhancers, etc., to ensure the expression of the iRNA intarget cells. Other aspects to consider for vectors and constructs arefurther described below.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-β-D1-thiogalactopyranoside (IPTG). A person skilled in the artwould be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an iRNA can be used. For example, a retroviral vectorcan be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)).These retroviral vectors contain the components necessary for thecorrect packaging of the viral genome and integration into the host cellDNA. The nucleic acid sequences encoding an iRNA are cloned into one ormore vectors, which facilitates delivery of the nucleic acid into apatient. More detail about retroviral vectors can be found, for example,in Boesen et al., Biotherapy 6:291-302 (1994), which describes the useof a retroviral vector to deliver the mdr1 gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993). Lentiviral vectors contemplated for useinclude, for example, the HIV based vectors described in U.S. Pat. Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated byreference.

Adenoviruses are also contemplated for use in delivery of iRNAs.Adenoviruses are especially attractive vehicles, e.g., for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walshet al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). In one embodiment, the iRNA can be expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector having, for example, either the U6 or H1 RNA promoters, orthe cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressingthe dsRNA featured in the invention, methods for constructing therecombinant AV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479;5,139,941; International Patent Application No. WO 94/13788; andInternational Patent Application No. WO 93/24641, the entire disclosuresof which are herein incorporated by reference.

Another typical viral vector is a pox virus such as a vaccinia virus,for example an attenuated vaccinia such as Modified Virus Ankara (MVA)or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

Pharmaceutical Compositions Containing Nucleic Acid Molecules (iRNA)

In one embodiment, the invention provides pharmaceutical compositionscontaining an iRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition containing the iRNAis useful for treating a disease or disorder related to the expressionor activity of a target gene. Such pharmaceutical compositions areformulated based on the mode of delivery. For example, compositions canbe formulated for systemic administration via parenteral delivery, e.g.,by intravenous (IV) delivery. In some embodiments, a compositionprovided herein (e.g., an LNP formulation) is formulated for intravenousdelivery. In some embodiments, a composition provided herein (e.g., acomposition comprising a GalNAc conjugate) is formulated forsubcutaneous delivery.

The pharmaceutical compositions featured herein are administered in adosage sufficient to inhibit expression of a target gene. In general, asuitable dose of iRNA will be in the range of 0.01 to 200.0 milligramsper kilogram body weight of the recipient per day, generally in therange of 1 to 50 mg per kilogram body weight per day. For example, thedsRNA can be administered at 0.05 μg/kg, 0.5 μg/kg, 1 μg/kg, 1.5 μg/kg,2 μg/kg, 3 μg/kg, 10 μg/kg, 20 μg/kg, 30 μg/kg, 40 μg/kg, or 50 μg/kgper single dose. The pharmaceutical composition may be administered oncedaily, or the iRNA may be administered as two, three, or more sub-dosesat appropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the iRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the iRNA over a several day period. Sustained releaseformulations are well known in the art and are particularly useful fordelivery of agents at a particular site, such as can be used with theagents of the present invention. In this embodiment, the dosage unitcontains a corresponding multiple of the daily dose.

The effect of a single dose on target gene levels can be long lasting,such that subsequent doses are administered at not more than 3, 4, or 5day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesrelated to target gene expression. Such models can be used for in vivotesting of iRNA, as well as for determining a therapeutically effectivedose and/or an effective dosing regimen.

A suitable mouse model is, for example, a mouse containing a transgeneexpressing huma target gene. Mice that have knock-in mutations can beused to determine the therapeutically effective dosage and/or durationof administration of target gene siRNA. The present invention alsoincludes pharmaceutical compositions and formulations that include theiRNA compounds featured in the invention. The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (e.g., by atransdermal patch), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal, oral or parenteral. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; subdermal, e.g., via an implanteddevice; or intracranial, e.g., by intraparenchymal, intrathecal orintraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue,such as a tissue that produces erythrocytes. For example, the iRNA canbe delivered to bone marrow, liver (e.g., hepatocyes of liver), lymphglands, spleen, lungs (e.g., pleura of lungs) or spine. In oneembodiment, the iRNA is delivered to bone marrow.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis Liposomes fall into two broadclasses. Cationic liposomes are positively charged liposomes whichinteract with the negatively charged DNA molecules to form a stablecomplex. The positively charged DNA/liposome complex binds to thenegatively charged cell surface and is internalized in an endosome. Dueto the acidic pH within the endosome, the liposomes are ruptured,releasing their contents into the cell cytoplasm (Wang et al., Biochem.Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C1215G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a target gene dsRNA is fully encapsulated in thelipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleicacid-lipid particle. As used herein, the term “SNALP” refers to a stablenucleic acid-lipid particle, including SPLP. As used herein, the term“SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNAencapsulated within a lipid vesicle. SNALPs and SPLPs typically containa cationic lipid, a non-cationic lipid, and a lipid that preventsaggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs andSPLPs are extremely useful for systemic applications, as they exhibitextended circulation lifetimes following intravenous (i.v.) injectionand accumulate at distal sites (e.g., sites physically separated fromthe administration site). SPLPs include “pSPLP,” which include anencapsulated condensing agent-nucleic acid complex as set forth in PCTPublication No. WO 00/03683. The particles of the present inventiontypically have a mean diameter of about 50 nm to about 150 nm, moretypically about 60 nm to about 130 nm, more typically about 70 nm toabout 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCTPublication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yeethylazanediyedidodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid may comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In some embodiments, the iRNA is formulated in a lipid nanoparticle(LNP).

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which is hereinincorporated by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are provided in thefollowing table.

TABLE 1 Examplary lipid formulations cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Cationic Lipid Lipid:siRNA ratio SNALP1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 S-XTC2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-C12-200/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:11-yl)ethylazanediyl)didodecan-2-ol (C12-200) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc- PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, and International Application No.PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated byreference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles featured in the invention may be preparedby known organic synthesis techniques, including the methods describedin more detail in the Examples. All substituents are as defined belowunless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x),—C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y),wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different andindependently hydrogen, alkyl or heterocycle, and each of said alkyl andheterocycle substituents may be further substituted with one or more ofoxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods featured in the invention may requirethe use of protecting groups. Protecting group methodology is well knownto those skilled in the art (see, for example, PROTECTIVE GROUPS INORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New YorkCity, 1999). Briefly, protecting groups within the context of thisinvention are any group that reduces or eliminates unwanted reactivityof a functional group. A protecting group can be added to a functionalgroup to mask its reactivity during certain reactions and then removedto reveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In one embodiments, nucleic acid-lipid particles featured in theinvention are formulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane). In general, thelipid of formula A above may be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R₃ and R₄ are independentlylower alkyl or R₃ and R₄ can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry

DCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H]-232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50mL). Organic phase was dried over an .Na2SO₄ and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO₄ then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9(×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6(×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+Calc. 654.6,Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations featured in the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

The compositions featured in the present invention may be formulatedinto any of many possible dosage forms such as, but not limited to,tablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. The compositions may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants may beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich NG., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials is also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310),hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants:

In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of iRNAs through the mucosa is enhanced.In addition to bile salts and fatty acids, these penetration enhancersinclude, for example, sodium lauryl sulfate, polyoxyethylene-9-laurylether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92); and perfluorochemical emulsions, such asFC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty Acids:

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

Bile Salts:

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents:

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofβ-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

Non-Chelating Non-Surfactants:

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers include, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass' D1Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or morebiologic agents which function by a non-RNAi mechanism. Examples of suchbiologic agents include agents that interfere with an interaction oftarget gene and at least one target gene binding partner.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are typical.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of diseases or disorders related totarget gene expression. In any event, the administering physician canadjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

Methods for Modulating Expression of a Target Gene

In yet another aspect, the invention provides a method for modulating(e.g., inhibiting or activating) the expression of a target gene, e.g.,in a cell or in a subject. In some embodiments, the cell is ex vivo, invitro, or in vivo. In some embodiments, the cell is an erythroid cell ora hepatocyte. In some embodiments, the cell is in a subject (e.g., amammal, such as, for example, a human) In some embodiments, the subject(e.g., the human) is at risk, or is diagnosed with a disease related totarget gene expression, as described above.

In one embodiment, the method includes contacting the cell with an iRNAas described herein, in an amount effective to decrease the expressionof a target gene in the cell. “Contacting,” as used herein, includesdirectly contacting a cell, as well as indirectly contacting a cell. Forexample, a cell within a subject (e.g., an erythroid cell or a livercell, such as a hepatocyte) may be contacted when a compositioncomprising an iRNA is administered (e.g., intravenously orsubcutaneously) to the subject.

The expression of a target gene may be assessed based on the level ofexpression of a target gene mRNA, a target gene protein, or the level ofa parameter functionally linked to the level of expression of a targetgene. In some embodiments, the expression of target gene is inhibited byat least 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95%. In someembodiments, the iRNA has an IC₅₀ in the range of 0.001-0.01 nM,0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM, 0.01-0.50 nM,0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, 0.01-10 nM. The IC₅₀ value maybe normalized relative to an appropriate control value, e.g., the IC₅₀of a non-targeting iRNA.

In some embodiments, the method includes introducing into the cell aniRNA as described herein and maintaining the cell for a time sufficientto obtain degradation of the mRNA transcript of a target gene, therebyinhibiting the expression of the target gene in the cell.

In one embodiment, the method includes administering a compositiondescribed herein, e.g., a composition comprising an iRNA that targetstarget gene, to the mammal such that expression of the target gene isdecreased, such as for an extended duration, e.g., at least two, three,four days or more, e.g., one week, two weeks, three weeks, or four weeksor longer. In some embodiments, the decrease in expression of targetgene is detectable within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours,or 24 hours of the first administration.

In another embodiment, the method includes administering a compositionas described herein to a mammal such that expression of the target geneis increased by e.g., at least 10% compared to an untreated animal. Insome embodiments, the activation of target gene occurs over an extendedduration, e.g., at least two, three, four days or more, e.g., one week,two weeks, three weeks, four weeks, or more. Without wishing to be boundby theory, an iRNA can activate target gene expression by stabilizingthe target gene mRNA transcript, interacting with a promoter in thegenome, and/or inhibiting an inhibitor of target gene expression.

The iRNAs useful for the methods and compositions featured in theinvention specifically target RNAs (primary or processed) of a targetgene. Compositions and methods for inhibiting the expression of a targetgene using iRNAs can be prepared and performed as described elsewhereherein.

In one embodiment, the method includes administering a compositioncontaining an iRNA, where the iRNA includes a nucleotide sequence thatis complementary to at least a part of an RNA transcript of the targetgene of the mammal to be treated. When the organism to be treated is amammal such as a human, the composition may be administered by any meansknown in the art including, but not limited to oral, intraperitoneal, orparenteral routes, including intracranial (e.g., intraventricular,intraparenchymal and intrathecal), intravenous, intramuscular,subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical(including buccal and sublingual) administration.

In certain embodiments, the compositions are administered by intravenousinfusion or injection. In some such embodiments, the compositionscomprise a lipid formulated siRNA (e.g., an LNP formulation, such as anLNP11 formulation as described herein) for intravenous infusion.

In other embodiments, the compositions are administered subcutaneously.In some such embodiments, the compositions comprise an iRNA conjugatedto a GalNAc ligand.

Target Genes and Methods for Treating Diseases Related to Expression ofa Target Gene

The dosing regimen and methods described herein can be used to inhibittarget gene expression and/or to treat a disease, a disorder, or apathological process that is related to target gene expression. Incertain embodiments, the target genes is chosen from: Factor VII, Eg5,PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, HAMP,Activated Protein C gene, Cyclin D gene, VEGF gene, antithrombin 3 gene,aminolevulinate synthase 1 gene, alpha-1-antitrypsin gene, tmprss6 gene,apoa1 gene, apoc3 gene, bc11a gene, klf gene, angpt13 gene, plk gene,PKN3 gene, HBV, HCV, p53 gene, angiopoietin gene, or angiopoietin-like 3gene. In certain embodiments, the target is chosen from: Eg5, PCSK9,TTR, HAMP, VEGF gene, antithrombin 3 gene, aminolevulinate synthase 1gene, alpha-1-antitrypsin gene, or tmprss6 gene.

TTR Target Gene

In one embodiment, the target gene is a TTR gene. Nucleic acid (e.g.,RNA) molecules capable of reducing expression of a TTR gene (e.g., toreduce TTR amyloid deposition, or treating a TTR-mediated amyloidosis(ATTR)) are described in, e.g., WO 2011/056883, the contents of whichare specifically incorporated by reference herein. In certainembodiments, the RNA molecule comprises an antisense strand comprising,or consisting of, 10, 15, 20, 25 or more contiguous nucleotidescomplementary to the transthyretin mRNA (e.g., wild type or mutant TTRmRNA e.g., V30M mutant TTR). In certain embodiments, the RNA moleculecomprises an antisense strand comprising, or consisting of, 10, 15, 20,25 or more contiguous nucleotides of an antisense oligonucleotidesequence disclosed in, e.g., WO 2011/056883, e.g., SEQ ID NOs: 170, 730,or 1010. In certain embodiments, the RNA molecule comprises an antisensestrand comprising, or consisting of, 10, 15, 20, 25 or more contiguousnucleotides of an antisense oligonucleotide sequence disclosed in, e.g.,WO 2011/056883, e.g., SEQ ID NOs: 170, 730, or 1010; and a sense stranddisclosed in, e.g., WO 2011/056883, e.g., SEQ ID NOs: 169, 729, or 1009.

PCSK9 Target Gene

In one embodiment, the target gene is a PCSK9 gene. Nucleic acid (e.g.,RNA) molecules capable of reducing expression of a PCSK9 gene (e.g., totreat a PCSK9-related disorder, e.g., lowering serum cholesterol) aredescribed, e.g., in WO 2012/05869, WO 2011/005861, WO 2011/028938, WO2010/148013, WO 2009/134487 and WO 2007/134161, the contents of whichare specifically incorporated by reference herein. In certainembodiments, the RNA molecule comprises an antisense strand comprising10, 15, 20, 25 or more contiguous nucleotides complementary to the PCSK9mRNA (e.g., wild type or mutant PCSK9 mRNA). In certain embodiments, theRNA molecule comprises an antisense strand comprising, or consisting of,10, 15, 20, 25 or more contiguous nucleotides of an antisenseoligonucleotide sequence disclosed, e.g., in WO 2012/05869, WO2011/005861, WO 2011/028938, WO 2010/148013, WO 2009/134487 and WO2007/134161. In certain embodiments, the RNA molecule comprises anantisense strand comprising, or consisting of, 10, 15, 20, 25 or morecontiguous nucleotides of an antisense oligonucleotide sequencedisclosed, e.g., in WO 2012/05869, WO 2011/005861, WO 2011/028938, WO2010/148013, WO 2009/134487 and WO 2007/134161; and a sense stranddisclosed, e.g., in WO 2012/05869, WO 2011/005861, WO 2011/028938, WO2010/148013, WO 2009/134487 and WO 2007/134161.

Eg5 Target Gene

In one embodiment, the target gene is an Eg5 gene. Nucleic acid (e.g.,RNA) molecules capable of reducing expression of an Eg5 gene (e.g., totreat an Eg5-related disorder) are described, e.g., in WO 2011/034798,WO 2010/105209, WO 2009/111658, and WO 2007/115168, the contents ofwhich are specifically incorporated by reference herein. In certainembodiments, the RNA molecule comprises an antisense strand comprising10, 15, 20, 25 or more contiguous nucleotides complementary to the Eg5mRNA (e.g., wild type or mutant Eg5 mRNA). In certain embodiments, theRNA molecule comprises an antisense strand comprising, or consisting of,10, 15, 20, 25 or more contiguous nucleotides of an antisenseoligonucleotide sequence disclosed, e.g., in WO 2011/034798, WO2010/105209, WO 2009/111658, and WO 2007/115168. In certain embodiments,the RNA molecule comprises an antisense strand comprising, or consistingof, 10, 15, 20, 25 or more contiguous nucleotides of an antisenseoligonucleotide sequence disclosed, e.g., in WO 2011/034798, WO2010/105209, WO 2009/111658, and WO 2007/115168; and a sense stranddisclosed, e.g., in WO 2011/034798, WO 2010/105209, WO 2009/111658, andWO 2007/115168.

VEGF Target Gene

In one embodiment, the target gene is a VEGF gene. Nucleic acid (e.g.,RNA) molecules capable of reducing expression of a VEGF gene (e.g., totreat a VEGF-related disorder) are described, e.g., in WO 2011/034798,WO 2010/105209, WO 2009/111658, and WO 2005/089224, the contents ofwhich are specifically incorporated by reference herein. In certainembodiments, the RNA molecule comprises an antisense strand comprising10, 15, 20, 25 or more contiguous nucleotides complementary to the VEGFmRNA (e.g., wild type or mutant VEGF mRNA). In certain embodiments, theRNA molecule comprises an antisense strand comprising, or consisting of,10, 15, 20, 25 or more contiguous nucleotides of an antisenseoligonucleotide sequence disclosed, e.g., in WO 2011/034798, WO2010/105209, WO 2009/111658, and WO 2005/089224. In certain embodiments,the RNA molecule comprises an antisense strand comprising, or consistingof, 10, 15, 20, 25 or more contiguous nucleotides of an antisenseoligonucleotide sequence disclosed, e.g., in WO 2011/034798, WO2010/105209, WO 2009/111658, and WO 2005/089224; and a sense stranddisclosed, e.g., in WO 2011/034798, WO 2010/105209, WO 2009/111658, andWO 2005/089224.

HAMP Target Gene

In one embodiment, the target gene is a Hepcidin Antimicrobial Peptide(HAMP) gene. Nucleic acid (e.g., RNA) molecules capable of reducingexpression of a HAMP gene (e.g., to treat a HAMP-related disorder, e.g.,a microbial infection) are described, e.g., in WO 2008/036933 and WO2012/177921, the contents of which are specifically incorporated byreference herein. In certain embodiments, the RNA molecule comprises anantisense strand comprising 10, 15, 20, 25 or more contiguousnucleotides complementary to the HAMP mRNA (e.g., wild type or mutantHAMP mRNA). In certain embodiments, the RNA molecule comprises anantisense strand comprising, or consisting of, 10, 15, 20, 25 or morecontiguous nucleotides of an antisense oligonucleotide sequencedisclosed, e.g., in WO 2008/036933 and WO 2012/177921. In certainembodiments, the RNA molecule comprises an antisense strand comprising,or consisting of, 10, 15, 20, 25 or more contiguous nucleotides of anantisense oligonucleotide sequence disclosed, e.g., in WO 2008/036933and WO 2012/177921; and a sense strand disclosed, e.g., in WO2008/036933 and WO 2012/177921.

TMPRSS6 Target Gene

In one embodiment, the target gene is a TMPRSS6 gene. Nucleic acid(e.g., RNA) molecules capable of reducing expression of a TMPRSS6 gene(e.g., to treat a TMPRSS6-related disorder) are described, e.g., in WO2012/135246, the contents of which are specifically incorporated byreference herein. In certain embodiments, the RNA molecule comprises anantisense strand comprising 10, 15, 20, 25 or more contiguousnucleotides complementary to the TMPRSS6 mRNA (e.g., wild type or mutantTMPRSS6 mRNA). In certain embodiments, the RNA molecule comprises anantisense strand comprising, or consisting of, 10, 15, 20, 25 or morecontiguous nucleotides of an antisense oligonucleotide sequencedisclosed, e.g., in WO 2012/135246. In certain embodiments, the RNAmolecule comprises an antisense strand comprising, or consisting of, 10,15, 20, 25 or more contiguous nucleotides of an antisenseoligonucleotide sequence disclosed, e.g., in WO 2012/135246; and a sensestrand disclosed, e.g., in WO 2012/135246.

5′-Aminolevulinic Acid Synthase 1 (ALAS1) Gene

In one embodiment, the target gene is an ALAS1 gene. Nucleic acid (e.g.,RNA) molecules capable of reducing expression of an ALAS1 gene (e.g., totreat an ALAS1-related disorder, e.g. a pathological processes involvingporphyrins or defects in the porphyrin pathway, such as, for example,porphyrias) are described, e.g., in U.S. Ser. No. 13/835,613, filed onMar. 15, 2013, the contents of which are specifically incorporated byreference herein. In certain embodiments, the RNA molecule comprises anantisense strand comprising 10, 15, 20, 25 or more contiguousnucleotides complementary to the ALAS1 mRNA (e.g., wild type or mutantALAS1 mRNA). In certain embodiments, the RNA molecule comprises anantisense strand comprising, or consisting of, 10, 15, 20, 25 or morecontiguous nucleotides of an antisense oligonucleotide sequencedisclosed, e.g., in U.S. Ser. No. 13/835,613. In certain embodiments,the RNA molecule comprises an antisense strand comprising, or consistingof, 10, 15, 20, 25 or more contiguous nucleotides of an antisenseoligonucleotide sequence disclosed, e.g., in U.S. Ser. No. 13/835,613;and a sense strand disclosed, e.g., in U.S. Ser. No. 13/835,613.

Complement Component 3 (C3) Gene

In one embodiment, the target gene is a Complement component 3 (C3)gene. Nucleic acid (e.g., RNA) molecules capable of reducing expressionof a C3 gene (e.g., to treat a C3-related disorder. C3 plays a centralrole in the complement system and contributes to innate immunity. Inhumans it is encoded on chromosome 19 by a gene called C3. In certainembodiments, the RNA molecule comprises an antisense strand comprising10, 15, 20, 25 or more contiguous nucleotides complementary to the C5mRNA (e.g., wild type or mutant C3 mRNA).

Complement Component 5 (C5) Gene

In one embodiment, the target gene is a Complement component 5 (C5)gene. Nucleic acid (e.g., RNA) molecules capable of reducing expressionof a C5 gene (e.g., to treat a C5-related disorder, e.g. a pathologicalprocesses involving inflammatory and cell killing processes. Thisprotein is composed of alpha and beta polypeptide chains that are linkedby a disulfide bridge. An activation peptide, CSa, which is ananaphylatoxin that possesses potent spasmogenic and chemotacticactivity, is derived from the alpha polypeptide via cleavage with aconvertase. In certain embodiments, the RNA molecule comprises anantisense strand comprising 10, 15, 20, 25 or more contiguousnucleotides complementary to the C5 mRNA (e.g., wild type or mutant C5mRNA).

As used herein, “a disorder related to target gene expression,” a“disease related to target gene expression, a “pathological processrelated to target gene expression,” or the like includes any condition,disorder, or disease in which target gene expression is altered (e.g.,elevated). For example, an iRNA targeting an ALAS1 target gene, or acombination thereof, may be used for treatment of conditions in whichlevels of a porphyrin or a porphyrin precursor (e.g., ALA or PBG) areelevated (e.g., certain porphyrias), or conditions in which there aredefects in the enzymes of the heme biosynthetic pathway (e.g., certainporphyrias). Disorders related to target gene expression include, forexample, X-linked sideroblastic anemia (XLSA), ALA deyhdratasedeficiency porphyria (Doss porphyria), acute intermittent porphyria(AIP), congenital erythropoietic porphyria, prophyria cutanea tarda,hereditary coproporphyria (coproporphyria), variegate porphyria,erythropoietic protoporphyria (EPP), and transient erythroporphyria ofinfancy.

As used herein, a “subject” to be treated according to the methodsdescribed herein, includes a human or non-human animal, e.g., a mammal.The mammal may be, for example, a rodent (e.g., a rat or mouse), aporcine, or a primate (e.g., a monkey). In some embodiments, the subjectis a human

In some embodiments, the subject is suffering from a disorder related totarget gene expression or is at risk of developing a disorder related totarget gene expression.

In some embodiments, an iRNA targeting target gene is administeredtogether with (e.g., before, after, or concurrent with) anothertreatment that may serve to alleviate one or more symptoms.

The term “decrease” (or “increase”) is intended to refer to a measurablechange, e.g., a statistically significant change. The change may be, forexample, at least 5%, 10%, 20%, 30%, 40%, 50% or more change (e.g.,decrease (or increase) relative to a reference value, e.g., a referencewhere no iRNA is provided).

The invention further relates to the use of an iRNA or a pharmaceuticalcomposition thereof, e.g., for treating a disorder related to targetgene expression, in combination with other pharmaceuticals and/or othertherapeutic methods, e.g., with known pharmaceuticals and/or knowntherapeutic methods, such as, for example, those which are currentlyemployed for treating the disorder.

The effective amount for the treatment of a disorder related to targetgene expression depends on the type of disorder to be treated, theseverity of the symptoms, the subject being treated, the sex, age andgeneral condition of the subject, the mode of administration and soforth. For any given case, an appropriate “effective amount” can bedetermined by one of ordinary skill in the art using routineexperimentation. It is within the ability of one skilled in the art tomonitor efficacy of treatment or prevention by measuring any one of suchparameters, or any combination of parameters. In connection with theadministration of an iRNA targeting target gene or pharmaceuticalcomposition thereof, “effective against” a disorder related to targetgene expression indicates that administration in a clinicallyappropriate manner results in a beneficial effect, e.g., for anindividual patient or for at least a fraction of patients, e.g., astatistically significant fraction of patients. Beneficial effectsinclude, e.g., prevention of or reduction of symptoms or other effects.For example, beneficial effects include, e.g., an improvement (e.g.,decrease in the severity or frequency) of symptoms, a reduction in theseverity or frequency of attacks, a reduced risk of developingassociated disease (e.g., neuropathy (e.g., progressive neuropathy),hepatocellular cancer), an improved ability to tolerate a precipitatingfactor, an improvement in quality of life, a reduction in the expressionof target gene, a reduction in a level (e.g., a plasma or urine level)of a marker of a disease or other effect generally recognized aspositive by medical doctors familiar with treating the particular typeof disorder.

A treatment or preventive effect is evident when there is animprovement, e.g., a statistically significant improvement in one ormore parameters of disease status, or by a failure to worsen or todevelop symptoms where they would otherwise be anticipated. As anexample, a favorable change of at least 10% in a measurable parameter ofdisease, e.g., at least 20%, 30%, 40%, 50% or more can be indicative ofeffective treatment. Efficacy for a given iRNA drug or formulation ofthat drug can also be judged using an experimental animal model for thegiven disease as known in the art. When using an experimental animalmodel, efficacy of treatment is evidenced when a statisticallysignificant reduction in a marker or symptom is observed.

Patients can be administered a therapeutic amount of iRNA. Thetherapeutic amount can be, e.g., 0.01-50 μg/kg, 0.01-10 μg/kg, 0.01-5μg/kg, 0.01-2 μg/kg, 0.01-1 μg/kg, and more typically, 0.01-0.5 mg, 0.1to 0.3 μg/kg. For example, the therapeutic amount can be 0.05, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, or 2.5, 3.0, 3.5, 4.0,4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg/kg dsRNA. Any of thesedosages can be used in the dosage regimens and methods described herein.

In some embodiments, the iRNA is formulated as a lipid formulation,e.g., an LNP formulation as described herein. In some such embodiments,the therapeutic amount is 0.01-5 μg/kg, e.g., 0.01, 0.02, 0.03, 0.04,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, or 5.0 μg/kg dsRNA. Any of these dosages can be usedin the dosage regimens and methods described herein.

In some embodiments, the lipid formulation, e.g., LNP formulation, isadministered intravenously.

In some embodiments, the iRNA is administered by intravenous infusionover a period of time, such as over a 5-minute, 10-minute, 15-minute,20-minute, 25-minute, 30-minute, 40-minute, 50-minute, 55-minute,60-minute, 65-minute, or 70-minute period.

In some embodiments, the iRNA is in the form of a GalNAc conjugate asdescribed herein. In some such embodiments, the therapeutic amount is0.5-50 μg/kg, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50μg/kg dsRNA. In some embodiments, the GalNAc conjugate is administeredsubcutaneously. Any of these dosages can be used in the dosage regimensand methods described herein.

In some embodiments, the administration is repeated, for example, on aregular basis, such as, daily, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration biweekly for three months,administration can be repeated once per month, for six months or a yearor longer.

In some embodiments, the iRNA agent is administered in two or more doses(e.g., two or more doses as described herein). In some embodiments, thefirst and second doses are adjusted such as to decrease ahypersensitivity response. In other embodiments, the number or amount ofsubsequent doses is dependent on the achievement of a desired effect,e.g., suppression of a target gene, or the achievement of a therapeuticor prophylactic effect, e.g., reduction or prevention of one or moresymptoms associated with a target gene disorder.

In some embodiments, the iRNA agent is administered according to aschedule. For example, the iRNA agent may be administered once per week,twice per week, three times per week, four times per week, or five timesper week. In some embodiments, the schedule involves regularly spacedadministrations, e.g., hourly, every four hours, every six hours, everyeight hours, every twelve hours, daily, every 2 days, every 3 days,every 4 days, every 5 days, weekly, biweekly, or monthly.

In some embodiments, the predetermined reduction is a decrease of atleast 10%, 20%, 30%, 40%, or 50% in target gene expression or symptoms(e.g., marker level).

In some embodiments, the predetermined reduction is a reduction of atleast 1, 2, 3, or more standard deviations, wherein the standarddeviation is determined based on the values from a reference sample,e.g., a reference sample as described herein.

Administration of the iRNA may reduce target gene mRNA or proteinlevels, e.g., in a cell, tissue, blood, urine or other compartment ofthe patient by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80% or at least 90% or more. Administration of the iRNA may reducelevels of products associated with target gene expression.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5%, 8% or 10% infusion dose, andmonitored for adverse effects, such as a hypersensivity or an allergicreaction, or for elevated lipid levels or blood pressure. In anotherexample, the patient can be monitored for unwanted effects.

Hypersensitivity Reactions and Biomarker Detection

A hypersensitivity reaction may occur as the doses of the compositionsdescribed herein are increased (e.g., at a dose of about 300 μg/kg). Inthose embodiments of increased doses, the subject can be treated withthe methods and dosage regiments described herein.

In some embodiments, the dosages and time periods of administration ofthe doses decribed herein are selected such that no substantial IRRand/or hypersensitivity reaction (e.g., a detectable IRR orhypersensitivity reaction) occurs in a subject. The hypersensitivityreaction can be an acute reaction. In some embodiments, the subject is ahuman. Alternatively, the subject can be an animal (e.g., an animalmodel for a complement or a hypersensitivity reaction (e.g., a porcinemodel as described herein and also in Szebeni et al. Nanomedicine. 2012February; 8(2):176-84; Szebeni et al. Adv Drug Deliv Rev. 2011 Sep. 16;63(12):1020-30 (2012); Szebeni et al. Adv Drug Delivery Rev)).

In some embodiments, the methods described herein do not cause adetectable hypersensitivity reaction, e.g., as measured by one or moreof the assays or symptoms described herein. In embodiments, the methodresults in a decrease in the hypersensitivity reaction, which is lessthan 1%, 5%, 10%, 25%, 30%, 35% or 40%, e.g., as measured by one or moreof of the assays or symptoms described herein. In certain embodiments,the changes described herein are compared to a reference parameter(e.g., a subject exposed to a bolus dose, or the subject prior totreatment).

In one embodiment, in response to the first and second dose regimendisclosed herein, the subject shows a reduced hypersensitivity reaction(e.g., a decreased hemodynamic change, relative to a reference parameter(e.g., a subject exposed to a bolus dose, or the subject prior totreatment).

In certain embodiments, the methods described herein cause a reducedhypersensitivity reaction, e.g., leading to a reduction (e.g., partialor complete reduction) in the administration of one or more of a steroid(e.g., dexamethasone or an equivalent), an analgesic (e.g.,paracetamol), or a histamine receptor antagonist (e.g., an H1 or an H2blocker). In other embodiments, the subject does not receiveadministration of a steroid (e.g., dexamethasone or an equivalent),within X hours of any of the initiation of administration of said firstdose, wherein X is less than 1 hour, 2 hours, 3 hours, 5 hours, 10hours, 15 hours, 24 hours or 48 hours.

Alternatively, or in combination, the subject may be medicated orpremedicated with one or more of a steroid, a histamine blocker oracetaminophen. For example, prior to (30 to 60 minutes), during, orafter administration of a dose as described herein (e.g., a first orsecond dose) of the compositions described herein, the subject canreceive one or more ofL a steroid (e.g., oral dexamethasone (8 mg) orintravenous dexamethasone (10 mg), or an equivalent), paracetamol oracetaminophen (e.g., 500 mg of oral paracetamol or an equivalent), ahistamine blocker (e.g., oral H2 blocker (e.g., ranitidine 150 mg orfamotidine 20 mg, or an equivalent) and/or an oral H1 blocker (e.g.,cetirizine (10 mg) or equivalent).

In certain embodiments, the method described herein further include thestep of evaluating the subject after administration of the first dose,the second dose, or both, for the presence of one or more of thefollowing: a skin reaction (e.g., urticaria, erythema, edema, rash,pruritus, eruptions), a hemodynamic change, e.g., a change in bloodpressure (e.g., hypotension or hypertension), a respiratory problem(e.g., laryngospasm, laryngeal edema, bronchospasm, dyspnea), pain(e.g., joint pain, back pain, abdominal pain or chest pain), or othermanifestations of hypersensitivity (e.g., one or more of fever, chills,nausea, vomiting or neurological changes).

Alternatively, or in combination, the methods described herein furtherinclude the step of evaluating the subject after administration of thefirst dose, the second dose, or both, for a change in a complementmarker, e.g., complement activation (e.g., a change in one or morecomplement factors chosen from Bb or C3a^(b)), wherein an increase thelevel of a complement biomarker is indicative of a hypersensitivityreaction.

A change in a complement marker can be detected in vivo or using an invitro assay. For example, a change in complement activation can bedetected using an assay that detects a complement cascade component,e.g, an assay (e.g., ELISA) that detects one or more of: totalcomplement proteins (e.g., C3 and C5); complement split products (e.g.,Bb, C3a or C5a), or terminal complement complement complex: sC5b-9.Additional examples of in vitro assays for evaluating complementactivity include CH₅₀: Residual total hemolytic complement activity, orAH50: Residual alternative pathway of hemolytic complement activity.Alternatively, a change in complement activation can be detected using acomplement depletion model (e.g., Cobra Venom Factor) forloss-of-function studies or a porcine model for complement-mediatedhypersensitivity (e.g., a porcine model as described herein). Inembodiments, a sample to be evaluated can be obtained from a subjectexposed to the compositions described herein. For example, the samplecan be a serum/plasma sample obtained for an in vivo assay. In otherembodiments, naïve serum/plasma can be used for modeling complementactivation in vitro.

Alternatively, or in combination, the methods described herein furtherinclude the step of evaluating the subject after administration of thefirst dose, the second dose, or both, for a change in thromboxanelevels, e.g., thromboxane B2 in plasma, e.g., wherein an increase in thelevel of thromboxane is indicative of an increased hypersensitivityreaction, e.g., an increased acute hypersensitivity reaction.

Alternatively, or in combination, the methods described herein furtherinclude the step of evaluating the subject after administration of thefirst dose, the second dose, or both, or changes in one or morecytokines chosen from interferon-alpha, interferon-gamma, tumor necrosisfactor-alpha, interleukin lbeta, interleukin 1 receptor antagonist(IL-1RA), interleukin-6, interleukin-8, interleukin-12, interleukin-18,interferon inducing protein-10, granulocyte colony stimulating factor.,or C-reactive protein (CRP). In certain embodiments, an increase in thelevel of IL-6, IL-8, IL-1RA or CRP is indicative of an increasedhypersensitivity reaction, e.g., relative to a reference parameter(e.g., a subject exposed to a bolus dose, or the subject prior totreatment).

Methods to measure polypeptide biomarkers (e.g., complement markers,thromboxane, cytokines), include, but are not limited to: Western blot,immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay(RIA), immunoprecipitation, surface plasmon resonance,chemiluminescence, fluorescent polarization, phosphorescence,immunohistochemical analysis, liquid chromatography mass spectrometry(LC-MS), matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy,fluorescence activated cell sorting (FACS), flow cytometry, laserscanning cytometry, hematology analyzer and assays based on a propertyof the protein including but not limited to DNA binding, ligand binding,or interaction with other protein partners.

The activity or level of a marker protein can also be detected and/orquantified by detecting or quantifying the expressed polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. These can include analyticbiochemical methods such as electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, immunohistochemistry and thelike. A skilled artisan can readily adapt known protein/antibodydetection methods for use in determining the expression level of one ormore biomarkers in a serum sample.

Another agent for detecting a polypeptide of the invention is anantibody capable of binding to a polypeptide corresponding to a markerof the invention, e.g., an antibody with a detectable label. Antibodiescan be polyclonal or monoclonal. An intact antibody, or a fragmentthereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”, withregard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin.

The polypeptide is detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

Marker expression level can also be assayed. Expression of a marker ofthe invention can be assessed by any of a wide variety of well knownmethods for detecting expression of a transcribed molecule or protein.Non-limiting examples of such methods include immunological methods fordetection of secreted, cell-surface, cytoplasmic, or nuclear proteins,protein purification methods, protein function or activity assays,nucleic acid hybridization methods, nucleic acid reverse transcriptionmethods, and nucleic acid amplification methods.

In certain embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g., mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. Marker expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

Methods of detecting and/or quantifying the gene transcript (mRNA orcDNA made therefrom) using nucleic acid hybridization techniques areknown to those of skill in the art (see e.g., Sambrook et al. supra).For example, one method for evaluating the presence, absence, orquantity of cDNA involves a Southern transfer as described above.Briefly, the mRNA is isolated (e.g., using an acidguanidinium-phenol-chloroform extraction method, Sambrook et al. supra.)and reverse transcribed to produce cDNA. The cDNA is then optionallydigested and run on a gel in buffer and transferred to membranes.Hybridization is then carried out using the nucleic acid probes specificfor the target cDNA.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1. siRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Oligonucleotide Synthesis.

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG,500A, Prime Synthesis) and RNA phosphoramidites with standard protectinggroups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeare purchased from (Promega). All phosphoramidites are used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichis used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes is used. The activator is 5-ethyl thiotetrazole(0.75M, American International Chemicals); for the PO-oxidationiodine/water/pyridine is used and for the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence is performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite are purchased from Biosearch Technologies. Conjugationof ligands to 5′-end and or internal position is achieved by usingappropriately protected ligand-phosphoramidite building block. Anextended 15 min coupling of 0.1 M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid-support-bound oligonucleotide. Oxidation of theinternucleotide phosphite to the phosphate is carried out using standardiodine-water as reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate is introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent. The cholesterol phosphoramidite is synthesized in house andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mLglass bottle (VWR). The oligonucleotide is cleaved from the support withsimultaneous deprotection of base and phosphate groups with 80 mL of amixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55°C. The bottle is cooled briefly on ice and then the ethanolic ammoniamixture is filtered into a new 250-mL bottle. The CPG is washed with2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixtureis then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dryice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionis then quenched with 50 mL of 20 mM sodium acetate and the pH isadjusted to 6.5. Oligonucleotide is stored in a freezer untilpurification.

Analysis

The oligonucleotides are analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phasepreparative HPLC. The unconjugated oligonucleotides are purified byanion-exchange HPLC on a TSK gel column packed in house. The buffers are20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodiumphosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides are pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotidess arediluted in water to 150 μL and then pipetted into special vials for CGEand LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

siRNA Preparation

For the general preparation of siRNA, equimolar amounts of sense andantisense strand are heated in 1×PBS at 95° C. for 5 min and slowlycooled to room temperature. Integrity of the duplex is confirmed by HPLCanalysis.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 2.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually inked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Absbeta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphateAfs 2′-fluoroadenosine-3′-phosphorothioate Asadenosine-3′-phosphorothioate C cytidine-3′-phosphate Cbbeta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate(Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Chds)2′-O-hexadecyl-cytidine-3′-phosphorothioate Cscytidine-3′-phosphorothioate G guanosine-3′-phosphate Gbbeta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioateGf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tb beta-L-thymidine-3′-phosphate Tbsbeta-L-thymidine-3′-phosphorothioate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Ubbeta-L-uridine-3′-phosphate Ubs beta-L-uridine-3′-phosphorothioate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate(Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Uhds)2′-O-hexadecyl-uridine-3′-phosphorothioate Usuridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate dA 2′-deoxyadenosine-3′-phosphatedAs 2′-deoxyadenosine-3′-phosphorothioate dC2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioatedG 2′-deoxyguanosine-3′-phosphate dGs2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine dTs2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine sphosphorothioate linkage L96¹N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3 (Aeo) 2′-O-methoxyethyladenosine-3′-phosphate (Aeos)2′-O-methoxyethyladenosine-3′-phosphorothioate (Geo)2′-O-methoxyethylguanosine-3′-phosphate (Geos)2′-O-methoxyethylguanosine-3′-phosphorothioate (Teo)2′-O-methoxyethyl-5-methyluridine-3′-phosphate (Teos)2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate (m5Ceo)2′-O-methoxyethyl-5-methylcytidine-3′-phosphate (m5Ceos)2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate ¹The chemicalstructure of L96 is as follows:

Example 2. Bolus Dosing Study of siRNA-LNP Formulation in a PorcineModel for Lipid Nanoparticle IRR/Hypersensitivity Reactions

This study evaluated the acute infusion-related reaction (IRR) followingintravenous (IV) bolus administration of siRNA-LNP to domestic Yorkshirepigs. Complement-mediated acute hypersensitivity reactions caused by asingle bolus dose of siRNA-LNP were investigated in this porcine model.Induction of tachyphylaxis by sequential repeat/increased bolus dosingof siRNA-LNP was also determined.

Methods and Experimental Design

Animals

The effect of siRNA-LNP on IRR was investigated in anesthetized,spontaneously breathing domestic pigs by administration of testformulation by IV bolus.

The porcine liposomal infusion reactions closely mimic human response.Administration of low (milligram) doses of nanoparticulate materials inpigs can lead to acute cardiopulmonary, hemodynamic, hematological,biochemical and dermatological changes within minutes, mimicking thehuman IRR to nanomedicines and biologics.

Exemplary features of porcine hypersensitivity reactions include, butnot limited to, cardiopulmonary distress (hyper/hypotension (pulmonaryhypertension with/without systemic hyper/hypotension), cardiacarrhythmias, tachycardia, bradycardia, cardiac arrest, decreased cardiacoutput, decreased pulse pressure, decreased left ventricularend-diastolic pressure), cutaneous changes (flushing, rash),bronchospasm, dyspnea/apnea, leucopenia/leukocytosis, thrombocytopenia,increased adenosine, and increased thromboxane A₂.

Pigs are sensitive to liposomes and are therefore a good model forhypersensitive human individuals. Exemplary porcine models ofcomplement-mediated infusion reactions are described, e.g., in SzebeniJ. et al., Adv. Drug Deliv. Rev. 2012; 64(15): 1706-1716, the content ofwhich is incorporated herein by reference.

Three male, domestic Yorkshire pigs (12-14 weeks/34-37 kg) were used inthis study. Animals were preanesthetized intramuscularly withCalypsol/Xilazine injections and the anesthesia was maintained usingisoflurane gas. Animals were breathing spontaneously. Respiration wasmonitored by using a pulse oximeter (fixed on the tongue), monitoringoxygen saturation and respiratory rate. Temperature was measured in therectum. A capnograph was connected to the tracheal tube to monitor etCO₂and the respiratory rate.

All incision areas were washed by a liberal application of povidoneiodine 10%.

The pigs were instrumented with Swan-Ganz catheter, introduced into thepulmonary artery through the right external jugular vein→rightatrium→right ventricle measuring the pulmonary arterial pressure (PAP);a pressure transducer was connected into the femoral artery to recordthe systemic arterial pressure (SAP). The left femoral vein wascannulated for blood sampling. The left external jugular vein was alsocannulated for the administration of test formulation and to maintain aslow drop infusion of saline (˜3 mL/kg/h).

ECG signals, according Einthoven's I—II-III leads were also continuouslyrecorded.

SiRNA-LNP Formulation

Each of the three male pigs received multiple IV bolus injections of thefollowing siRNA-LNP formulation: MC3/DSPC/Chol/PEG2000-C-DMG(50/10/38.5/1.5 mol %). The ratio of total lipid/siRNA: 12.23. The siRNAduplex has the following sequences: GccuGGAGuuuAuucGGAAdTsdT (SEQ ID NO:5) (sense strand) and UUCCGAAuAAACUCcAGGCdTsdT (SEQ ID NO: 6) (antisensestrand). This siRNA duplex targets rat proprotein convertasesubtilisin/kexin type 9 (PCSK9) gene and has no detectablecross-reactivity with pig sequence.

Administration

Each animal received fixed unit sequential doses of 0 mg (salinecontrol), 2 mg, 2 mg, 10 mg, and 20 mg (all doses based on siRNAcontent) by IV bolus injection via cannulated left jugular vein (washedin by 5 mL saline), with at least 0.5 hr between doses to allow forrecovery to baseline. Dosing solutions were prepared from a 2 mg/mLstock solution diluted in 0.9% sterile saline to appropriateconcentrations for a constant total injection volume of 10 mL/injection.Saline served also as negative control, each experiment started with 10mL bolus injection of saline to test the reactivity. Following the lastinjection of the tested siRNA-LNP the animals received an IV bolusinjection of Zymosan (0.5 μg/kg), which is known to induce acutecardiovascular changes associated with complement activation. Table 3shows the lipid dose rates and total doses used in this study.

TABLE 3 Lipid Dose Rates and Total Doses Sequential Bolus Doses Bolus 1Bolus 2 Bolus 3 Bolus 4 Total Lipid Dose/Bolus (mg) 24.5 24.5 122.3244.6 Cumulative Lipid Dose (mg) 24.5 49.0 171.3 416.0 LipidConcentration (mg/ml) 2.5 2.5 12.2 24.5 Lipid Dose Rate (mg/min) 49 49244 489 Dosing Time 0.5 min (estimate)

Data Collection

The following parameters were evaluated: cardiovascular (pulmonary andsystemic pressure, heart rate), ECG (Einthoven's I—II-III leads),respiratory parameters (respiratory rate, end-tidal CO2, blood 02saturation), clinical signs, hematology (platelets (PLT), white bloodcells (WBC), red blood cells (RBC), hemoglobin content or RBCs (Hb),lymphocytes (LYM), and granulocytes (GR)), and body temperature.

Plasma samples were collected pre- and post-each dose at specifictime-points for evaluation of Thromboxane B2 for pig 3 only. Serum andplasma samples were collected pre- and post-each dose at specifictime-points for evaluation of biomarkers.

Results

IV bolus administration of the test siRNA-LNP resulted in an acuteinfusion-related reaction (IRR) in all three animals following the firstdose of the formulation (2 mg).

In the first animal (pig 1), the pulmonary arterial pressure (PAP)increased to 370% of the pre-test value combined with an increase insystemic arterial pressure (SAP) to 130% and a minor change in heartrate (HR) to 107% of the pre-test value, respectively. FIG. 1 showschanges in absolute values, i.e., PAP, SAP are expressed in mmHg, HR inbeat/min, in pig 1.

In the second animal (pig 2), a sharp PAP increase to 500% of thepre-test value was observed, while the SAP showed an initial moderateincrease to 107% that was immediately followed by a sharp decrease to28% of the SAP pre-test value, combined with a decrease in HR to 63%.FIG. 2 shows changes in absolute values, i.e., PAP, SAP are expressed inmmHg, HR in beat/min, in pig 2.

In the third animal (pig 3), a sharp increase in PAP to 404% of thepre-test value was observed, while the SAP showed an initial increase to110% followed by a sharp decrease to 43% and the HR showed an initialincrease to 120% followed by a sharp decrease to 68% of the pre-testvalues, respectively.

In animals two and three, skin changes (whole body flush and abdominalrash) were also observed. Respiratory arrest occurred in animals two andthree and both animals were manually resuscitated and received aninjection of epinephrine.

The second 2 mg siRNA-LNP dose evoked negligible changes in all 3animals, suggesting that the animals had become tachyphylactic toadministration of an equivalent dose.

Administration of a subsequent 10 mg dose caused mild PAP increasewithout SAP or HR changes in pigs 1 and 2, and only a negligible PAPincrease and SAP decrease in pig 3. The subsequent 20 mg dose evokedcardiovascular reactions in all three animals that displayed slowerkinetics and milder responses than what was observed after the firstdose: pig 1, long lasting, biphasic increases in PAP; pig 2, shorterchanges in PAP, SAP and HR were observed; pig3, mild increases in PAPand steady decrease in SAP decrease.

For pig 3, the plasma samples were analyzed for Thromboxane B2 (TXB2)levels by ELISA. After the first test article dose a sharp increase inTXB2 of −58-fold over baseline was observed 2 min. post dose thatdecreased to −10-fold over baseline at 30 min post dose. The subsequentdoses did not result in TXB2 release.

The vehicle injection (saline solution, 10 mL IV bolus), that precededin every experiment the test material injections did not evoke anycardiovascular alterations.

Conclusion

A 2 mg (based on siRNA) unit IV bolus dose (10 mL) of siRNA-LNPformulation resulted in a significant IRR in all animals based oncardiovascular changes (PAP, SAP, HR) and clinical signs of toxicity in2 out of 3 animals. Repeated administration of the same dose after a1-1.25 hr recovery period did not result in a similar reaction,suggesting that the animals were refractory to further stimulation.Subsequent administration of a 5-fold higher dose (10 mg) resulted in amild cardiovascular reaction in 2 out of 3 animals and the kinetics ofthese responses were slower than what was observed following the firstdose of 2 mg. A stronger response was observed in all animals after anadditional 20 mg dose of the formulation. These data suggest that thetachyphylaxis observed after the first and second doses of 2 mg could beovercome by administration of a higher dose. However, none of theresponses to the higher doses displayed the same rapid kinetics andseverity that was seen for the first dose.

Example 3: Microdosing Study of siRNA-LNP Formulation in a Porcine Modelfor Lipid Nanoparticle Hypersensitivity Reactions (Protocol I)

This study evaluated the acute and delayed infusion-related reaction(IRR) following intravenous (IV) infusion at different doserates/regimens of siRNA-LNP to domestic Yorkshire pigs.

Methods and Experimental Design

Animals

The same animal model described in Example 2 was used in this study. Theanimals were prepared as described in Example 2. In addition, for urinesamples a short midline incision was made in the abdomen, and a plastictube was inserted and fixed into the urinary bladder.

SiRNA-LNP Formulation

The siRNA-LNP formulation used in this study is described in Example 2.

Administration

Six (6) pigs were administered a total dose of 0.5 μg/kg siRNA-LNPformulation by IV infusion at different dose rates/regimens (2animals/group). The dosing regimen was as follows:

-   -   pigs 01 and 02:0.5 μg/kg infused over 60 minutes    -   pigs 03 and 04:0.5 μg/kg infused over 120 minutes    -   pigs 05 and 06:0.5 μg/kg total dose, 1/10^(th) of the dose        infused over 15 minutes followed immediately by the remaining        9/10^(t)h of the dose over 60 min.

Dosing solutions were prepared from a 1.98 mg/mL stock solution dilutedin 0.9% sterile saline to appropriate concentrations for a constanttotal infusion volume of 50 mL. At the end of the cardiovascularmonitoring period, the animals received an IV bolus injection of Zymosan(0.5 μg/kg), which is known to induce acute cardiovascular changesassociated with complement activation.

Data Collection

The following parameters were evaluated: cardiovascular (pulmonary andsystemic pressure, heart rate), ECG (Einthoven's I—II-III leads),respiratory parameters (respiratory rate, end-tidal CO₂, blood O₂saturation), clinical signs, blood cell and platelet counts, plasmahistamine, plasma tryptase, and body temperature. Plasma and urinesamples were collected before infusion and during and after the infusionat specific timepoints for evaluation of Thromboxane B2. Serum andplasma samples were collected at specific timepoints for evaluation ofbiomarkers. Biomarkers included plasma Thromboxane B2 and serumcytokines/chemokines (GM-CSF, IFNγ, IL-10, IL-12, IL-18, IL-1RA, IL-1α,IL-1β, IL-2, IL-4, IL-6, IL-8, and TNFα).

Results

IV infusion of 0.5 μg/kg siRNA-LNP formulation over 60 minutes in pig01and pig02 resulted in mild cardiovascular changes during the infusionand severe changes 15-30 min after the infusion in both animals.

During infusion, in pig01 only a slow, moderate increase in pulmonaryarterial pressure (PAP; 190% of the pre-test value) was observed, whilein pig02 only a mild increase in systemic arterial pressure (SAP) wasobserved in the first 5 min that was followed by a gradual decrease inSAP (78% of the pre-test value). After the infusion, dramatic elevationsin PAP, SAP and heart rate (HR) were observed in both animals combinedwith skin changes (pig01: whole body flush and rash; pig02: whole bodyflush) and ECG changes. For pig01, the PAP had increased to 430% of thepre-test value at 150 min post start of infusion and started to declineat 175 min, but never returned to the pre-test value. HR and SAPfollowed similar kinetics. For pig02, a similar reaction was observedbut the changes started around 15 min later than in pig01 and the PAPreached a maximum of 382% of the pre-test value at 140 min post start ofinfusion. FIG. 3 shows changes in absolute values, i.e., PAP, SAP areexpressed in mmHg, HR in beat/min, in pig01.

IV infusion of 0.5 μg/kg the tested siRNA-LNP formulation over 120minutes in pig03 and pig04 resulted in mild cardiovascular changesduring the infusion and severe changes after the infusion in bothanimals. Within 30 min after the infusion ended the PAP started to risesharply. The PAP reached a maximum of 625% of the pre-test value at 285min post start of infusion in pig03 and 278% at 240 min post start ofinfusion in pig04, respectively. In both animals mild skin flushing andECG changes were observed.

IV infusion of 0.5 μg/kg the tested siRNA-LNP formulation in two stepsby first infusing 1/10^(th) of the dose over 15 min immediately followedby 9/10^(th) of the dose over 60 minutes in pig05 and pig06 resulted inonly very mild changes overall. During the entire infusion period

PAP, SAP and HR were stable for pig05 and there were only minimal PAPfluctuations between 66-136% of the pre-test value for pig06. FIG. 4shows changes in absolute values, i.e., PAP, SAP are expressed in mmHg,HR in beat/min, in pig06. After the infusion, only a mild, gradualincrease in PAP to a maximum of 160% at 160 min post start of infusionwas observed in pig05. In pig05 no skin or ECG changes were observed andin pig06 no skin changes and only minimal ECG changes were seen.

For pig01 and pig02, Thromboxane B2 (TxB2) plasma levels remained stableduring IV infusion. After end of infusion, an increase in TxB2 over timewas observed for both animals, with pig01 reaching a maximum of 5-foldover TxB2 baseline level at 240 min and pig02 reaching a maximum of16-fold over baseline at 150 min. TxB2 release coincided with PAPincrease. Urine TxB2 levels were analyzed only for pig01, but all valueswere above ULOQ of the assay.

In pig03 and pig04 TxB2 plasma also increased over time and maximumlevels of 6.5-fold over baseline at 240 min and 4.7-fold over baselinewas reached at 225 min. TxB2 release coincided with PAP increase.

In pig05 TxB2 plasma levels were high at baseline and decreased 0.3-foldover time. In pig06, TxB2 increased over time and reached a maximum of4.6-fold over baseline at 150 min, which is similar in magnitude to pigs01, 03, and 04, but there was no corresponding increase in PAP.

For serum cytokines, an increase in the levels of IL-6, IL-8, IL-18 andIL-1RA were observed. No significant changes were observed for GM-CSF,IFNγ, IL-10, IL-12, IL-1α, IL-1β, IL-2, IL-4, and TNFα.

Conclusion

None of the infusion regimens used in this study resulted in an acuteIRR similar to the reaction that was observed within minutes of a bolusadministration of 2 mg of the tested siRNA-LNP formulation as describedin Example 1.

Administration of 0.5 μg/kg the tested siRNA-LNP formulation by IVinfusion over 60 min or 120 min resulted in a severe response thatstarted within 30 min after end of infusion and was characterized bypulmonary hypertension, systemic hypo- or hypertension, skin flushingand ECG alterations. Microdosing 1/10^(th) of the dose followed by9/10^(th) of the dose did not result in a significant response at any ofthe times observed.

Overall, the two animals within each of the three dose groups showedresponses with similar kinetics but different intensities. This is inagreement with the response heterogeneity seen in Example 1. There was aclear difference between the single step infusions when compared to themicrodosed group, since there were no severe changes at any time in thelatter group. These data suggest microdosing as a valuable strategy forpreventing lipid-nanoparticle induced IRR.

In pigs, 01, 02, 03, 04, and 06 rises of plasma TxB over time wereobserved. The TxB2 in pig06 from the microdosed group was in a similarrange of fold-induction over baseline as the 60/120 min infusion groupand the TxB2 levels in pig05 from the microdosed group had high baselinelevels.

Example 4: Microdosing Study of siRNA-LNP Formulation in a Porcine Modelfor Lipid Nanoparticle Hypersensitivity Reactions (Protocol II)

This study uses an alternative microdosing protocol to evaluate theacute and/delayed infusion-related reaction (IRR) following intravenous(IV) infusion at different dose rates/regimens of siRNA-LNP to domesticYorkshire pigs.

Methods and Experimental Design Animals The effect of microdosing ofsiRNA-LNP formulation on preventing or reducing IRR was investigatedusing the animal model as described in Example 2.

SiRNA-LNP Formulation

The siRNA-LNP formulation used in this study is described in Example 2.

Administration

Six (6) pigs are administered a total dose of 5.85 μg/kg testedsiRNA-LNP formulation by IV infusion at different dose rates/regimens (3animals/group). The dosing regimen is shown in Table 4.

TABLE 4 Study Design Test Total No. Article/ Total Dose Dose of PositiveDose Volume Rate Group Pigs Control (mg/kg) (mL) (mg/min) Route 1 3siRNA- 5.85 50 10 IV Infusion LNP Zymosan 5 5 N/A IV Bolus 2 3 siRNA-5.85 50 1 IV Infusion LNP (for first 15 min) 10 (for remainder of dose)Zymosan 5 5 N/A IV Bolus

The total dose (5.85 μg/kg) and the total infusion volume (50 mL) areheld constant for each animal. Based on individual animal body weight,an appropriate volume of stock siRNA-LNP formulation is diluted with0.9% saline to a total volume of 60 mL in a sterile glass vial. At theend of the cardiovascular monitoring period, the animals received an IVbolus injection of Zymosan (0.5 μg/kg), which is known to induce acutecardiovascular changes associated with complement activation.

Data Evaluation

Data are collected and analyzed as described in Examples 2 and 3.

Experiments conducted as described in Example 4 demonstrated a cleardifference between the microdosed group (1 mg/min, followed by 10mg/min) and the straight 10 mg/min group. The TxB2 elevations weresignificantly higher in the 10 mg/min group.

Example 5: Microdosing Study of siRNA-LNP Formulation in Humans

This study evaluated the infusion-related reaction (IRR) followingintravenous (IV) infusion at different dose rates/regimens of siRNA-LNPin human patients.

Patients were administered a total dose of 0.30 μg/kg siRNA-LNPformulation by IV infusion at different dose rates/regimens. The totaldose was based on the amount of siRNA in the formulation. The lipid:siRNA ratio ranged from 11.5-14:1. For certain batch, it was around11.6:1.

The dosing regimen was as follows:

-   -   Ten (10) patients: 0.30 μg/kg infused over a period of 60        minutes. The 60-minute infusion occurred at a rate of 3 mL/min        for the full 60 minutes.    -   Thirty-two (32) patients: 0.30 μg/kg infused over a period of 70        minutes (microdosing regimen). The 70-minute infusion occurred        at a rate of 1 mL/min for the first 15 minutes and 3 mL/min for        the remainder of the time.

Patients were assessed for symptoms, including blood pressure, heartrate and body temperature. FIG. 5 shows the percentages of dosesassociated with infusion-related reactions (IRRs) in those. As shown inFIG. 5, the incidence of IRRs was decreased from 15% ( 3/20) in patientswho received the 60-minute regimen to 2% (2/102) in patients whoreceived the 70-minute microdosing regimen.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of reducing an infusion-related response (IRR), or ahypersensitivity reaction, or both, in a subject, to a compositioncomprising a lipid formulation and a nucleic acid molecule, said methodcomprising administering to a subject: a first dose of said composition;a second dose of said composition; and wherein one, two, three, four,five, six or all of a)-g) of the following conditions is met: a) theamount of said composition administered in said first dose is no morethan 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50,of the amount of said composition administered in said second dose; b)the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40or 50, of the total amount of said composition administered; c) thefirst dose is administered over a time period that is no more than 1/X,wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time period over whichthe second dose is administered; d) the rate of administration of saidfirst dose is no more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40 or 50, of the rate of administration of said second dose;e) the amount of said composition administered in said first dose is nomore than 20, 30 or 40 μg, nucleic acids per kg body weight, and thesecond dose is greater than said first dose; f) the amount of saidcomposition administered in said second dose is greater than 100, 200 or300 μg nucleic acids per kg body weight, and the second dose is greaterthan said first dose; or g) the dosages and time periods ofadministration of said first and second doses are selected such that nosubstantial IRR and/or hypersensitivity reaction occurs in said subject.2. A method of reducing the expression of a target gene, or treating adisorder related to the target gene, in a subject, the methodcomprising: administering to the subject a first dose and a second doseof a composition, said composition comprising a lipid formulation and anucleic acid molecule, wherein said first and second doses areadministered in an amount sufficient to reduce expression of a targetgene, or treat the disorder, in the subject; and wherein one, two,three, four, five or all of a)-f) of the following conditions is met: a)the amount of said composition administered in said first dose is nomore than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40or 50, of the amount of said composition administered in said seconddose; b) the amount of said composition administered in said first doseis no more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,30, 40 or 50, of the total amount of said composition administered; c)the first dose is administered over a time period that is no more than1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time period overwhich the second dose is administered; d) the rate of administration,e.g., in mg/min or mL/min, of said first dose is no more than 1/X,wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50, of therate of administration of said second dose; e) the amount of saidcomposition administered in said first dose is no more than 20, 30 or 40μg, nucleic acids per kg body weight, and the second dose is greaterthan said first dose; or f) the amount of said composition administeredin said second dose is greater than 100, 200 or 300 μg nucleic acids perkg body weight, and the second dose is greater than said first dose. 3.The method of either of claim 1, wherein: (i) the rate of administrationof said first dose is one or more of: about 5% to 50%, about 5% to about20%, about 5% to about 10%, about 10% to about 40%, about 10% to about20%, or about 20% to about 40% of the rate of administration of thesecond dose; (ii) the total amount of said composition administered insaid first dose is chosen from between (and including): about 0.5% to20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%,about 2% to about 10%, about 2% to about 5%, or about 5% to about 10% ofthe total amount of said composition, or the amount of the compositionadministered in said second dose; or (iii) both (i) and (ii). 4.-6.(canceled)
 7. The method of claim 1, wherein: (i) the rate ofadministration of the lipid formulation in said first dose is chosenfrom about 0.05 μg/min/kg to about 50 μg/min/kg, about 0.1 μg/min/kg toabout 25 μg/min/kg, about 1 μg/min/kg to about 15 μg/min/kg, or about 5μg/min/kg to about 10 μg/min/kg; (ii) the rate of administration of thelipid formulation in said second dose is chosen from about 0.5 μg/min/kgto about 500 μg/min/kg, about 1 μg/min/kg to about 250 μg/min/kg, about10 μg/min/kg to about 150 μg/min/kg, or about 50 μg/min/kg to about 100μg/min/kg; or (iii) both (i) and (ii), optionally, wherein the firstdose is administered over a time period that is no greater than 1/X,wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 the time period over which thetotal dose is administered. 8.-10. (canceled)
 11. The method of eitherof claim 1, wherein: (i) the rate of administration of the nucleic acidmolecule in said first dose is chosen from about 0.01 μg/min/kg to about5 μg/min/kg, about 0.02 μg/min/kg to about 2.5 μg/min/kg, about 0.05μg/min/kg to about 1 μg/min/kg, or about 0.1 μg/min/kg to about 0.5μg/min/kg; (ii) the rate of administration of the nucleic acid moleculein said second dose is chosen from about 0.1 μg/min/kg to about 50μg/min/kg, about 0.2 μg/min/kg to about 25 μg/min/kg, about 0.5μg/min/kg to about 10 μg/min/kg, or about 1 μg/min/kg to about 5μg/min/kg; or (iii) both (i) and (ii), optionally, wherein the firstdose is administered over a time period that is no greater than 1/X,wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 the time period over which thetotal dose is administered. 12.-15. (canceled)
 16. The method of eitherof claim 1, wherein: (i) the amount of lipid formulation administered insaid first dose is chosen from about 0.5 μg/kg to about 1000 μg/min/kg,about 1 μg/min/kg to about 500 μg/kg, about 10 μg/kg to about 250 μg/kg,or about 50 μg/kg to about 150 μg/kg; (ii) the amount of lipidformulation administered in said second dose is chosen from: about 20μg/kg to about 50000 μg/kg, about 100 μg/kg to about 25000 μg/kg, about500 μg/kg to about 10000 μg/kg, or about 1000 μg/kg to about 5000 μg/kg;or (iii) both (i) and (ii), optionally, wherein the first dose isadministered over a time period that is no greater than 1/X, whereinX=2, 3, 4, 5, 6, 7, 8, 9 or 10 the time period over which the total doseis administered. 17.-19. (canceled)
 20. The method of either of claim 1,wherein: (i) the amount of the nucleic acid molecule in said first doseis chosen from about 0.1 μg/kg to about 20 μg/kg, about 0.25 μg/kg toabout 15 μg/kg, about 0.5 μg/kg to about 10 μg/kg, or about 1 μg/min/kgto about 7.5 μg/kg; (ii) the amount of the nucleic acid molecule in saidsecond dose is chosen from about 2 μg/kg to about 1000 μg/kg, about 5μg/kg to about 750 μg/kg, about 10 μg/kg to about 500 μg/kg, or about 50μg/kg to about 300 μg/kg; or (iii) both (i) and (ii), optionally,wherein the first dose is administered over a time period that is nogreater than 1/X, wherein X=2, 3, 4, 5, 6, 7, 8, 9 or 10 the time periodover which the total dose is administered.
 21. The method of claim 1,wherein: (i) the second dose is administered over a time period that isat least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times greater than the time periodover which the first dose is administered; (ii) the first dose isadministered over a time period that is no greater than 1/X, whereinX=2, 3, 4, 5, 6, 7, 8, 9 or 10 the time period over which the total doseis administered; (iii) the first dose is administered over a time periodthat is between 5% and 50%, between 10% and 45%, between 15% and 40%,between 20% and 35%, or between 25% and 30% of the time period ofadministration of the second dose; (iv) the first dose is administeredover a time period that is between 5 minutes and 60 minutes, between 10minutes and 50 minutes, between 20 minutes and 40 minutes, between 5minutes and 30 minutes, or between 10 minutes and 20 minutes; (v) thesecond dose is administered over a time period that is between 30minutes and 180 minutes, between 40 minutes and 120 minutes, between 45minutes and 90 minutes, or between 50 minutes and 65 minutes; or (vi) nomore than 1, 10, 20, 30, 60, or 180 minutes separates the completion ofthe administration of the first dose and the initiation of theadministration of the second dose. 22.-27. (canceled)
 28. The method ofclaim 1, further comprising administering to the subject one or moreadditional doses of the composition.
 29. The method of claim 1, whereinsaid first and second doses are administered by intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion, intravenously by infusion, at a substantially constant ratevia a pump or a sustained or controlled release formulation, or as agradient or multiple rates. 30.-32. (canceled)
 33. The method of claim29, wherein: (i) the flow rate of administration of the first dose is nomore than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40or 50, of the flow rate of administration of said second dose; (ii) theflow rate of administration of the first dose is chosen from about 0.5to 1.5 mL/min, about 0.8 to 1.3 mL/min, about 1 to 1.2 mL/min, about 1mL/min or 1.1 mL/min; (iii) the flow rate of administration of thesecond dose is chosen from about 2 to 4 mL/min, about 2.5 to 3.7 mL/min,about 3 to 3.5 mL/min, about 3 mL/min or 3.3 mL/min; (iv) both (ii) and(iii); or (v) the total volume of infusion is about 100 to 300 mL, about150 to 250 mL, about 180 mL or 200 mL. 34.-37. (canceled)
 38. The methodof claim 1, wherein the IRR and/or hypersensitivity reaction is an acutehypersensitivity reaction during dose administration or occurs afteradministration of the second dose is completed.
 39. (canceled)
 40. Themethod of claim 1, further comprising evaluating the subject afteradministration of the first dose, the second dose, or both, for one ormore of the following: (i) the presence of one or more of the following:a skin reaction, a hemodynamic change, a change in blood pressure, arespiratory problem, pain, or one or more of fever, chills, nausea,vomiting or neurological changes; (ii) a change in a complementbiomarker chosen from one or more of complement activation, or a changein one or more complement factors chosen from Bb or C3a^(b), wherein anincrease the level of a complement biomarker is indicative of an IRRand/or hypersensitivity reaction; (iii) a change in thromboxane levelsor thromboxane B2 in plasma, wherein an increase in the level ofthromboxane or thromboxane B2 is indicative of an increasedhypersensitivity reaction; or (iv) a change in one or more cytokineschosen from interferon-alpha, interferon-gamma, or tumor necrosisfactor-alpha, interleukin lbeta, interleukin 1 receptor antagonist(IL-1RA), interleukin-6, interleukin-8, interleukin-12, interleukin-18,interferon inducing protein-10, granulocyte colony stimulating factor,or C-reactive protein (CRP), wherein an increase in the level of IL-6,IL-8, IL-1RA or CRP is indicative of an increased hypersensitivityreaction. 41.-44. (canceled)
 45. The method of claim 1, wherein: (i)said method does not cause a detectable IRR and/or hypersensitivityreaction; (ii) said method results in a decrease in the IRR and/orhypersensitivity reaction, which is less than 1%, 5%, 10%, 25%, 30%, 35%or 40%; (iii) said method causes a reduced IRR and/or hypersensitivityreaction leading to a reduction in the administration of one or more ofa steroid, an analgesic, or a histamine receptor antagonist; (iv) saidsubject does not receive administration of a steroid within X hours ofany of the initiation of administration of said first dose, wherein X isless than 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 24hours or 48 hours; or (v) if said first and second dose regimen isprovided to a porcine subject, the subject will show a reduced IRRand/or hypersensitivity reaction, relative to a subject exposed to abolus dose, or the subject prior to treatment. 46.-53. (canceled) 54.The method of claim 1, wherein the lipid is a cationic or a non-cationiclipid, or a combination thereof.
 55. The method of claim 54, wherein thecationic lipid is chosen from N,N-dioleyl-N,N-dimethylammonium chloride(DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP,also referred to as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniumchloride in U.S. Pat. No. 8,158,601),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA,also referred to as N-(2,3-dioleyloxy)propyl-N,N-N-triethylammoniumchloride in U.S. Pat. No. 8,158,601),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), bis(3-pentyloctyl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate,1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin M-C3-DMA, MC3, or M-C3),1,1′-(2-(4-(24(2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof; and/or wherein the cationic lipidcomprises from about 20 mol % to about 60 mol %, or about 40 mol % ofthe total lipid present in the formulation. 56.-93. (canceled)
 94. Themethod of claim 1, wherein the nucleic acid molecule is chosen from: adouble stranded RNA (dsRNA) molecule, a single-stranded RNAi molecule, amicroRNA (miRNA), an antisense RNA, a short hairpin RNA (shRNA), anantagomir, an mRNA, a decoy RNA, a DNA, a plasmid, or an aptamer.95.-96. (canceled) 97.-114. (canceled)
 115. A kit for administration ofa first dose and a second dose of a composition, comprising: providing acomposition, said composition comprising a lipid formulation and anucleic acid molecule, wherein said second amount is greater than saidfirst amount; and instruction for administration, wherein the first doseis instructed to be administered over a time period that is no more than1/X, wherein X is 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the time period overwhich the second dose is administered; and the rate of administration ofsaid first dose is no more than 1/X, wherein X is 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40 or 50, of the rate of administration of saidsecond dose.
 116. A method of preparing the first and second dose of thekit of claim 115, comprising: modifying the rate of administration ofthe composition, such that the dose is adjusted.
 117. (canceled) 118.The method of claim 1, wherein: (i) the first dose is administered at afirst nucleic acid dose rate between 1.5 and 2 μg/kg/min, and the seconddose is administered at a second nucleic acid dose rate between 4 and 6μg/kg/min; (ii) the first dose is administered at a first lipid doserate between 15 and 25 μg/kg/min, and the second dose is administered ata second lipid dose rate between 55 and 75 μg/kg/min; (iii) both (i) and(ii). 119.-120. (canceled)
 121. The method of claim 1, wherein: (i) thefirst dose is administered at between 0.5 and 1.5 mL/min, and the seconddose is administered at between 2.5 and 3.5 mL/min; (ii) the first doseis administered over a period of between 10 and 20 minutes, and thesecond dose is administered over a period of between 50 and 60 minutes;or (iii) both (i) and (ii).
 122. (canceled)
 123. The method of claim 1,wherein: (i) the first nucleic acid dose is between 20 and 30 μg/kg, andthe second nucleic acid dose is between 250 and 300 μg/kg; (ii) thefirst lipid dose is between 250 and 400 μs/kg, and the second lipid doseis between 2500 and 4000 μs/kg; or (iii) both (i) and (ii). 124.(canceled)
 125. The method of claim 1, wherein: (i) the total nucleicacid dose in the first and the second doses is between 0.2 and 0.4μg/kg; (ii) the total lipid dose in the first and the second doses isbetween 3.0 and 4.5 μg/kg; or (iii) both (i) and (ii).
 126. (canceled)127. The method of claim 1, wherein: (i) the first nucleic acid doserate is between 0.1 and 0.15 mg/min, and the second nucleic acid doserate is between 0.3 and 0.4 mg/min; (ii) the first lipid dose rate isbetween 1.0 and 1.5 mg/min, and the second lipid dose rate is between3.5 and 4.5 mg/min; or (iii) both (i) and (ii).
 128. (canceled)
 129. Themethod of claim 1, wherein: (i) the first nucleic acid dose is between1.5 and 2.0 mg and the second nucleic acid dose is between 15 and 25 mg;(ii) the first lipid dose is between 15 and 25 mg, and the second lipiddose is between 200 and 300 mg; or (iii) both (i) and (ii). 130.-147.(canceled)